WO2024259624A1

WO2024259624A1 - A modified rsv f protein, a nanoparticle, a composition and a vaccine against respiratory syncytial virus infection

Info

Publication number: WO2024259624A1
Application number: PCT/CN2023/101642
Authority: WO
Inventors: Hongliang LIV; Xiaolin Liu
Original assignee: Shenzhen Genius Biotech Service Co. Ltd
Priority date: 2023-06-21
Filing date: 2023-06-21
Publication date: 2024-12-26

Abstract

The present invention provides a modified RSV F protein, a nanoparticle, a composition and a vaccine adapted for mucosal administration, and in particular for intranasal administration. Intranasal administration of the modified RSV F protein, the nanoparticle, the composition and the vaccine can elicit mucosal and systemic antiviral immunity, resulting in the reduced virus-associated pathology and reduced viral burdens, thereby preventing, ameliorating and/or treating disease caused by infection of the RSV virus.

Description

A Modified RSV F Protein, A Nanoparticle, A Composition and A Vaccine Against Respiratory Syncytial Virus Infection

TECHNICAL FIELD

The present invention relates to a modified RSV (Respiratory Syncytial Virus) F protein, a nanoparticle, a composition and a vaccine adapted for mucosal administration, and in particular for intranasal administration.

BACKGROUND

Human respiratory syncytial virus (RSV) is the leading viral agent of severe acute respiratory infections in infants and young children worldwide. Every year, RSV is responsible for an estimated 118,200 deaths worldwide among children <5 years of age, with 99%of these deaths occurring in developing countries. RSV disease and the associated economic burden also are substantial in developed countries. While severe RSV disease has been commonly thought to occur predominantly in young infants <6 months of age, it was recently recognized that >50%of hospitalizations and in-hospital deaths of children with RSV occur among infants ≥6 months of age. Thus, RSV morbidity and mortality are frequent throughout infancy and young childhood. Infants at high risk for severe RSV disease due to premature birth or underlying illness can receive passive immunoprophylaxis with a monoclonal antibody against RSV called palivizumab, with substantial protective efficacy. However, this is not indicated for infants in general and is not cost-effective for use in resource-limited settings. A pediatric RSV vaccine is needed to reduce the morbidity and mortality associated with RSV infection. Despite decades of effort and recent progress, a licensed RSV vaccine is not yet available.

RSV is an enveloped virus with a non-segmented negative-sense RNA genome of approximately 15,200 nucleotides. The genome is expressed as 10 separate mRNAs encoding 11 proteins. The 3’ to 5’ gene order (identified by encoded proteins) is as follows: nonstructural protein 1 (NS1) , NS2, nucleocapsid protein (N) , phosphoprotein (P) , matrix protein (M) , small hydrophobic protein (SH) , attachment protein (G) , fusion protein (F) , RNA synthesis factors M2-1 and M2-2 (encoded by overlapping open reading frames [ORFs] in the M2 mRNA) , and polymerase protein (L) ; there also are short leader and trailer regions at the 3’ and 5’ genome ends, respectively. The RSV G protein is the major viral attachment protein. RSV F mediates fusion of the viral envelope with the cellular membrane during viral entry and may also have attachment activity. The RSV F and G proteins are the two RSV neutralization antigens and the major protective antigens. F is generally considered to be a more potent neutralization and protective antigen than G, and its amino acid sequence is much more conserved among RSV strains. RSV F is produced in a prefusion (pre-F) conformation that is metastable and can be readily triggered to undergo a major irreversible conformational rearrangement that drives membrane fusion and leaves F in a highly stable post-fusion (post-F) conformation. Pre-F and post-F share some neutralizing epitopes, but most of the neutralizing activity in convalescent human sera recognizes epitopes specific to pre-F. RSV F can be substantially stabilized in the pre-F conformation by structure-based engineering, such as by the introduction of a disulfide bond called DS and two hydrophobic cavity-filling amino acid substitutions called Cav1. DS-Cav1-stabilized pre-F is substantially more immunogenic in rodents and nonhuman primates than post-F either as a subunit vaccine or expressed by a bacteria vector.

Development of a pediatric RSV vaccine has been challenging. Inactivated RSV and purified RSV subunit vaccines are associated with enhancement of RSV disease in RSV-naive young children and experimental animals, respectively, and are contraindicated for administration to RSV-naive infants and young children (although they appear to be safe in RSV-experienced recipients) . In contrast, these presently are being developed as candidate pediatric RSV vaccines for intranasal (i. n. ) administration are not associated with priming for enhanced RSV disease.

Current RSV candidate vaccines consist of either inactivated whole virus, recombinant subunit vaccine, virus vector attenuated vaccine or mRNA vaccine. Fusion proteins are the antigens to which protective antibody responses are directed, fusion protein being the major protective antigen. Estimates of the efficacy of these parenterally administered vaccines vary greatly. Such vaccines are believed to act primarily by eliciting circulating anti-RSV IgG antibodies that transudate into the lower respiratory tract.

Both secretory IgA and serum IgG participate in immunity to RSV virus. Moreover, in cotton rats, a number of published studies have demonstrated the importance of respiratory IgA to protection against RSV infection. It has also been found that an advantage of stimulating a local IgA response to RSV is that it is often of a broader specificity than the serum response and thus can provide cross-protection against viruses possessing RSV F molecules different from those present in the vaccine. Accordingly, RSV vaccines that elicit both local secretory and serum anti-RSV F responses should provide superior immunity to current vaccines. However, parenteral vaccination (intramuscular, sub-cutaneous etc., ) is not effective at eliciting local antibody production, if there has been no previous mucosal exposure (e.g. infection) . In order to stimulate the mucosal immune system, the vaccine must be applied topically to a mucosal surface.

Mucosal administration of vaccine would have a number of advantages over traditional parenteral immunization regimes. Paramount amongst these is more effective stimulation of the local mucosal immune system of the respiratory tract and the likelihood that vaccine uptake rates would be increased because the fear and discomfort associated with injections would be avoided. Accordingly, a number of attempts have been made to develop mucosal vaccines. A drawback however is that subunit vaccines are often poorly immunogenic when given mucosally. In order to overcome this problem, different approaches to improving the immunogenicity of RSV vaccines given orally or intranasally have included the use of the B sub-unit of cholera toxin (CTB) as an adjuvant, encapsulation of the vaccine in a variety of microspheres, and the use of live attenuated strains. To date however no practical means of enhancing the immunogenicity of mucosally administered vaccines has been developed.

SUMMARY OF THE INVENTION

To this end, the present invention provides a modified RSV F protein, a nanoparticle, a composition and a vaccine adapted for mucosal administration, and in particular for intranasal administration. Intranasal administration of the modified RSV F protein, the nanoparticle, the composition and the vaccine elicits mucosal and systemic antiviral immunity, resulting in the reduced virus-associated pathology and reduced viral burdens.

In the first aspect, the present invention provides a modified RSV F protein, wherein the modified RSV F protein comprises an amino acid sequence having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.

In some embodiments, the modified RSV F protein further comprises an inactivated primary fusion cleavage site.

In some embodiments, the inactivated primary fusion cleavage site is obtained by mutation of arginine residues at positions 133, 135, and 136 of SEQ ID NO: 1 to glutamine.

In some embodiments, the modified RSV F protein comprises or consists of SEQ ID NO: 5.

In some embodiments, the modified RSV F protein is a monomeric RSV F protein.

In the second aspect, the present invention provides a nucleic sequence encoding the modified RSV F protein according to the first aspect.

In the third aspect, the present invention provides a cell comprising the nucleic sequence according to the second aspect.

In some embodiments, the cell is E. coli DH5α cell.

In some embodiments, the E. coli DH5α cell is named as pCBS220-sF/DH5ɑ, classified as E. coli DH5α engineering strain of recombinant human respiratory syncytial virus fusion protein F, has been deposited with China General Microbiological Culture Collection Center (CGMCC) (No. 1, West Beichen Rd., Chaoyang District, Beijing 100101, China) under the accession number CGMCC No. 25524 since Aug. 12, 2022.

In the fourth aspect, the present invention provides a nanoparticle comprising a viral protein and at least one polymer (s) , wherein the viral protein consists of RSV antigen or antigens, wherein the RSV antigen contains the modified RSV F protein according to the first aspect.

In some embodiments, the nanoparticle is immunogenic.

In some embodiments, the nanoparticle is a RSV antigen or antigens entrapped within the nanoparticle.

In some embodiments, the polymer is a water soluble, non-adhesive polymer.

In some embodiments, at least one polymer is selected from the group consisting of poly (lactic-co-glycolic acid) , polyethylene glycol, polyethylene oxide, polyalkylene glycol, polyalkylene oxide and polyethylene glycol-poly (lactic-co-glycolic acid) polymer.

In some embodiments, the polymer is poly (lactic-co-glycolic acid) (PLA) .

In some embodiments, the nanoparticle has an average diameter of about 250 nm to about 600 nm as measured by dynamic light scattering.

In the fifth aspect, the present invention provides a composition comprising: (i) the nanoparticle according to according to the second aspect; and (ii) an adjuvant.

In some embodiments, the adjuvant is a Mycobacterium lysate.

In some embodiments, the adjuvant is a Mycobacterium tuberculosis whole cell lysate.

In the sixth aspect, the present invention provides a vaccine comprising the composition according to the fifth aspect and a pharmaceutically acceptable carrier.

In the seventh aspect, the present invention provides a method for preventing, ameliorating and/or treating disease caused by infection of the RSV virus comprising administering the effective amount of the composition according to the fifth aspect or the vaccine according to the sixth aspect to a subject in need thereof.

In the eighth aspect, the present invention provides a method for eliciting an immune response comprising administering the effective amount of the composition according to the fifth aspect or the vaccine according to the sixth aspect to a subject in need thereof.

In some embodiments, the composition or vaccine is mucosally administered.

In some embodiments, the composition or vaccine is administered to mucosal surface.

In some embodiments, mucosal surface is selected from the group consisting of intratracheal mucosal surface, intranasal mucosal surface, rectal mucosal surface and vaginal mucosal surface.

In some embodiments, the subject is a human.

In some embodiments, the subject is a child with <5 years of age.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows PAGE Blue staining results of purified F protein, F monomers, Trimers F.

Fig. 2 shows negative stain electron microscopy of RAg.

Fig. 3 shows morphology of RAgs entrapped PLGA nanoparticles.

Fig. 4 shows r F protein release kinetics from PLGA. Data shown is the cumulative mass fraction of F protein released from PLGA nanoparticles. Data represent mean±SEM. Results are representative of three independent experiments with duplicate samples.

Fig. 5A shows the relative expression of the inflammatory cytokine IL-8 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 5B shows the relative expression of the inflammatory cytokine IL-12 p40 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 5C shows the concentration of the inflammatory cytokine INF-γ in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 5D shows the concentration of the inflammatory cytokine IL-1β in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 5E shows the concentration of the inflammatory cytokine IL-6 in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 5F shows the concentration of the inflammatory cytokine TNF-α in the Monocyte-derived dendritic cells stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 6A shows the concentration of the inflammatory cytokine INF-γ in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 6B shows the concentration of the inflammatory cytokine IL-1β in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 6C shows the concentration of the inflammatory cytokine IL-6 in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 6D shows the concentration of the inflammatory cytokine TNF-α in the alveolar macrophages stimulated with PLGA nanoparticles or RSV-F loaded nanoparticles.

Fig. 7 shows that the stained results of lung tissues which were collected from individual cotton rat (n = 6 per group) . Lung tissue sections were stained with hematoxylin and eosin (H&E) to assess inflammation. (A) PLGA+WCL (control) ; (B) RSV-F+WCL (I.N. ) ; (C) RSV-F-NP (I. N) suspension; (D) RSV-F-NP+WCL (I.N. ) .

Fig. 8 shows the reduced viral Burden in the lungs of vaccinated animals.

Fig. 9A shows the increased RSV specific IgA in the nasal fluid.

Fig. 9B shows the increased RSV specific IgA in the BAL fluid. Nasal fluid was collected on days 0, 14 and 28 post-vaccination, and on days 3 and 6 post-challenge. BAL fluid was collected during necropsy on day 7 post-challenge. The samples were diluted 1: 2. Indirect ELISAs were used to quantify BRSV-, F-protein specific IgA in (A) nasal fluid and (B) BAL fluid. Results represent n=6 animals/group. Data represent mean±SEM. *p<0.05 **p<0.01 compared to the PLGA+WCL group.

Fig. 10A shows the enhanced RSV-Specific T cell Response in the Peripheral blood.

Fig. 10B shows the concentration of the inflammatory cytokine IFNγ secreted by PBMCs from the BRSV-F nanovaccine-administrated animals in response to whole virus.

Fig. 10C shows the concentration of the inflammatory cytokine IL-17A secreted by PBMCs from the BRSV-F nanovaccine-administrated animals in response to whole virus.

DETAILED DESCRIPTION OF THE INVENTION

It should be noted that, unless defined otherwise, all technical or scientific terms used in one or more embodiments of the present specification shall have the meaning commonly understood by a person skilled in the art to which the invention belongs.

As used herein, the abbreviation “RAg” stands for recombinant F (rF) . The recombinant F (rF) comprises one immunogenic RSV proteins and therefore the recombinant F (rF) can be considered a subunit antigen.

As used herein, the abbreviation “NP-RAg” stands for nanoparticle-recombinant antigen. This represents the nanoparticle encapsulated recombinant rF.

As used herein, “treatment” or “treating, ” or “alleviating” or “ameliorating” are used interchangeably herein. These terms refers to an approach for obtaining beneficial or desired results including but not limited to therapeutic benefit. By “therapeutic benefit” is meant eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved by eradicating or ameliorating one or more of the physiological symptoms associated with the underlying disorder such that amelioration is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder.

As used herein, “prevention” and “preventing” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a prophylactic benefit.

As used herein, “individual” “or “subject” are used interchangeably and include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and most preferably humans. The combinations of the invention can be administered as described herein to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like) , farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, and the like) . In some embodiments, the mammal treated in the methods of the invention is a child with <5 years of age.

An effective amount of a disclosed composition or the vaccine required for use in therapy varies with the nature of the condition being treated, the length of treatment time desired, the age and the condition of the subejct, and is ultimately determined by the attending physician. In general, however, doses employed for infants and young children treatment typically are in the range of about one or more doses of composition or the vaccine described herein. The desired dose may be conveniently administered in a single dose consisting of between about 100 μg and about 150 μg of composition or the vaccine described herein.

Described herein is a modified RSV F protein, wherein the modified RSV F protein comprises an amino acid sequence having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.

In some embodiments, the modified RSV F protein comprises or consists of SEQ ID NO: 5. Over time, small amount of truncated RSV F peptide may arise due to proteolysis. Advantageously, however, the modified RSV F protein disclosed herein minimizes such degradation and provides extended stability.

In some embodiments, the modified RSV F protein is a monomeric RSV F protein.

Described herein is a nucleic sequence encoding the modified RSV F protein according to the present invention.

Described herein is a cell, wherein the cell comprises the nucleic sequence according to the present invention.

In some embodiments, the cell is E. coli DH5α cell.

In some embodiments, the E. coli DH5α cell has been deposited with China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 25524.

Described herein is a nanoparticle, wherein nanoparticle comprises a viral protein and at least one polymer (s) , wherein the viral protein consists of RSV antigen or antigens, wherein the RSV antigen contains the modified RSV F protein according to the present invention.

In some embodiments, an RSV F nanoparticle comprises one or more the modified RSV F protein monomer encapsulated with PLGA. The RSV F nanoparticle, the nanoparticle has an average diameter of about 200 nm to about 600 nm as measured by dynamic light scattering. In some embodiments of the RSV F nanoparticle, each RSV F protein monomer contains an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1. In some embodiments of the RSV F nanoparticle, each RSV F protein monomer contains an RSV F protein selected from the group consisting of RSV F proteins having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1 and an inactivated primary fusion cleavage site.

The modified RSV F protein induces the production of neutralizing antibodies. In further embodiments, the neutralizing antibodies recognize the modified RSV F protein in a post-fusion state and/or a pre-fusion state.

Examples of nanoparticles include, but are not limited to nanoparticles composed of one or more polymers. In some embodiments, the one or more polymers is/are a water soluble, non-adhesive polymer. In some embodiments, polymer is polyethylene glycol (PEG) or polyethylene oxide (PEO) . In some embodiments, the polymer is polyalkylene glycol or polyalkylene oxide. In some embodiments, the one or more polymers is/are a biodegradable polymer. In some embodiments, the one or more polymers is/are a biocompatible polymer that is a conjugate of a water soluble, non-adhesive polymer and a biodegradable polymer. In some embodiments, the biodegradable polymer is poly (lactic-co-glycolic acid) (PLGA) . In some embodiments, the nanoparticle is composed of PEG-PLGA polymers.

In some embodiments, the nanoparticle is formed by self-assembly. Self-assembly refers to the process of the formation of a nanoparticle using components that will orient themselves in a predictable manner forming nanoparticle predictably and reproducably. In some embodiments, the nanoparticles are formed using amphiphillic biomaterials which orient themselves with respect to one another to form nanoparticles of predictable dimension, constituents, and placement of constituents.

In some embodiments, the nanoparticle has a positive zeta potential. In some embodiments, the nanoparticle has a net positive charge at neutral pH. In some embodiments, the nanoparticle comprises one or more amine moieties at its surface. In some embodiments, the amine moiety is a primary, secondary, tertiary, or quaternary amine. In some embodiments, the amine moiety is an aliphatic amine. In some embodiments, the nanoparticle comprises an amine-containing polymer. In some embodiments, the nanoparticle comprises an amine-containing lipid. In some embodiments, the nanoparticle comprises a protein or a peptide that is positively charged at neutral pH. In some embodiments, the nanocarrier is a latex particle. In some embodiments, the nanoparticle with the one or more amine moieties on its surface has a net positive charge at neutral pH.

In some embodiments, depending on the ratio of PLGA used for the polymerization, different forms of PLGA can be obtained and utilized. These forms are typically identified in regard to the monomers' ratio used. Additional examples of suitable nanoparticles include chitosan, calcium phosphate, lipids of various bacteria like E. Coli, mycobactera, leptospira and mixtures thereof. In one example, the nanoparticle can be derived mixing about 180 mg of PLGA to about 5 mg of RAg (or about 36 mg PLGA to 1 mg RAg) . The entrapment (encapsulation) efficiency of RAg can vary. In one embodiment, the nanoparticle was 50-55%entrapped/encapsulated, calculated based on amount of total RSV protein used in the entrapment. Entrapped recombinant RAg can be administered as mixtures of entrapped/encapsulated and unentrapped/unencapsulated antigen or the entrapped/encapsulated antigens can be further purified.

Nanoparticles can aid the delivery of the recombinant RAg and/or can also be immunogenic. Delivery can be to a particular site of interest, e.g. the mucosa. In some embodiments, the nanoparticle can create a timed release of the RAg to enhance and/or extend the immune response. In some embodiments, the nanoparticle is associated with the RAg such that the composition can elicit an immune response. The association can be, for example, wherein the nanoparticle is coupled or conjugated with the RAg. By coupled and conjugated is meant that there is a chemical Linkage between the nanoparticle and the RAg. In some embodiments, the recombinant RAg is entrapped or encapsulated within the nanoparticle. In some embodiments, the RAg is entrapped within the nanoparticle by a water/oil/water emulsion method.

In some embodiments, an RSV F nanoparticle comprises one or more RSV F protein monomer encapsulated with PLGA. The RSV F nanoparticle, the nanoparticle has an average diameter of about 250 nm to about 600 nm as measured by dynamic light scattering.

In some embodiments, a method of manufacturing an RSV F protein nanoparticle comprises preparing an RSV F protein extract from a host cell. In some embodiments of the method, the weight ratio of PLGA: RSV F protein is about 180 to about 5.

In one aspect, the disclosure provides compositions containing RSV viral protein nanoparticles. In particular aspects, the RSV proteins are recombinantly expressed in a suitable host cell. In one embodiment, the host cell is an bacteria cell. In an exemplary embodiment, the bacteria cell is a DH5α cell.

In some embodiments of the composition, the composition comprises: (i) the nanoparticle according to the present invention; and (ii) an effective amount of adjuvant.

An adjuvant component can increase the strength and/or duration of an immune response to an antigen relative to that elicited by the antigen alone. A desired functional characteristic of an adjuvant component is its ability to enhance an appropriate immune response to a target antigen.

The adjuvant can be any composition, pharmacological or immunological agent that modifies the effect of other agents, such as the antigens described herein. Examples of adjuvants include, but are not limited to Mycobacterium lysate (including a Mycobacterium tuberculosis whole cell lysate) , a Mycobacterium smegmatis (including Mycobacterium smegmatis whole cell lysate) , cholera toxin B subunit, and E. coli heat labile mutant toxin. Other examples of adjuvants include evolutionarily conserved molecules, so called PAMPs, which include liposomes, lipopolysaccharide (LPS) , molecular cages for antigen, components of bacterial cell walls, and endocytosed nucleic acids such as double-stranded RNA (dsRNA) , single-stranded DNA (ssDNA) , and unmethylated CpG dinucleotide-containing DNA. Additional examples of adjuvants, include, but are not limited to aluminum containing adjuvants that include a suspension of minerals (or mineral salts, such as aluminum hydroxide, aluminum phosphate, aluminum hydroxyphosphate) onto which antigen is adsorbed. Adjuvants Additional examples of adjuvants, include, but are not limited to aluminum- (alum-) free adjuvants, which are formulated in the absence of any such aluminum salts. Alum-free adjuvants include oil and water emulsions, such as water-in-oil, and oil-in-water (and variants thereof, including double emulsions and reversible emulsions) , liposaccharides, lipopolysaccharides, immunostimulatory nucleic acids (such as CpG oligonucleotides) , liposomes, Toll-like Receptor agonists (particularly, TLR2, TLR4, TLR7/8 and TLR9 agonists) , and various combinations of such components.

In particular embodiments, the disclosure provides immunogenic compositions comprising one or more viral protein species in a nanoparticle structure where the protein is in the form of a monomer and each nanoparticle contains at least one monomer entrapped with PLGA. In particular aspects, a nanoparticle consists of an antigen, such as a viral protein, from only one pathogen.

Disclosed herein is a vaccine, wherein the vaccine comprises the composition and a pharmaceutically acceptable carrier.

For RSV vaccine, in some embodiments, the RSV vaccine comprises one RSV F nanoparticle wherein each nanoparticle comprises at least one RSV F protein monomer surrounded by PLGA.

The carrier may be any suitable carrier known to a person skilled in the art, for example a protein or an antigen presenting cell, such as a dendritic cell (DC) . Carrier proteins include keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid. Alternatively, the carrier protein may be tetanus toxoid or diphtheria toxoid. Alternatively, the carrier may be a dextran such as sepharose. The carrier must be physiologically acceptable to humans and safe.

The immunogenic composition and vaccine disclosed herein are suitable for preventing, ameliorating and/or treating disease caused by infection of the RSV virus.

Disclosed herein is a method for preventing, ameliorating and/or treating disease caused by infection of the RSV virus, wherein the method comprises administering the effective amount of the composition or the vaccine to a subject in need thereof.

Disclosed herein is a method for eliciting an immune response, wherein the method comprises administering the effective amount of the composition or the vaccine to a subject in need thereof.

In some embodiments, a method of preventing, ameliorating and/or treating disease caused by infection of the RSV virus or for eliciting an immune response comprises administering one or more doses of the vaccine composition. In some embodiments of the method, each dose consists of between about 100 μg and about 150 μg of the protein antigen.

In some embodiments, the composition or vaccine is mucosally administered.

In some embodiments, the subject is a human.

In some embodiments, the subject is a child with <5 years of age.

Disclosed herein are nanoparticles for inducing immune responses, methods for producing and administering mucosally them and composition or vaccine containing them. The nanoparticle provides antigen surrounded with PLGA that result in a structure that provides enhanced the stimulation of a protective IgA mucosal immune response and an IgG systemic immune response. In addition, the nanoparticles offer especially good antigen presentation to immune systems.

The present invention is further illustrated by the following examples that, however, are not to be construed as limiting the scope of protection. The features disclosed in the foregoing description and in the following examples may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof.

Example 1 Preparation of the recombinant F (rF)

In this example, a plasmid with the DNA encoding the vaccine candidate was prepared, amplified and purified from E. coli cells and were transformed. The protein was expressed and purified from cell fermentation.

The amino acids were shown below with features coded as indicated.

The amino acid sequence of the RSV F protein was shown as SEQ ID NO: 1:

The amino acid sequence of the signal peptide was shown as SEQ ID NO: 2

The amino acid sequence of the TEV cleavage site was shown as SEQ ID NO: 3

The amino acid sequence of the solubility enhancer 6×his Tag (italics, bold) was shown as SEQ ID NO: 4:

The amino acid sequence of the modified RSV F protein was shown as SEQ ID NO: 5:

Materials, Methods and Results

1.1 Cloning, Expression and Purification of F Immunogen

To generate our F-based vaccine candidate, we used the sequence of strain of RSV (GenBank: KY249682.1) . A DNA fragment encoding the following elements were synthesized by Twist Biosciences and cloned into bacteria expression vector pCBS220 to generate pRSV-F: (1) A ETB secretory signal peptide (SEQ ID NO: 2) , (2) F protein (SEQ ID NO: 1) , (3) C-terminal flanking TEV cleavage site (SEQ ID NO: 3) , and (4) three CR blocks (ENLYFQSLCRCRCRCR (SEQ ID NO: 4) ) followed by a six-Histidine tag (SEQ ID NO: 5) . The additional amino acid residues flanking the N-and C-terminal ends of the F were added for restriction sites (Hind III/Age I/Kpn I and Xma I/Nsi I, respectively) to facilitate transfer of the gene into different expression vectors. Following cleavage of the signal peptide, the C-terminal E residue is expected to remain on the final antigen. Additional non-coding sequences were used at the ends of the gene to ensure optimal Kozak sequence and compatibility with the plasmid pCBS220. The final construct was sequenced to confirm intactness of the F gene.

The plasmid pCBS220-F was transformed into E. coli DH5α (Bio-RAD) according to the manufacturer’s instructions. The designated pCBS220-sF/DH5ɑ has been deposited with China General Microbiological Culture Collection Center (CGMCC) on August 12, 2022 under the accession number CGMCC No. 25524.

The resulting strains were tested first at the shake flask scale. Induced bands of the expected size for processed and unprocessed (56 kDa and 180 kDa, respectively) were detected by SDS-PAGE. About half of the protein was processed (indicating localization to the periplasm) , and of the processed about half was in the soluble fraction and half in the insoluble fraction. Expression studies were scaled up to 20-L bioreactors. Densitometry of the Coomassie-stained SDS-PAGE gels showed that 18%of the total RSV F produced was processed and soluble. The strain produced 3.2 g/L of all forms of RSV F processed and soluble was 0.6 g/L. Recombinant F was isolated from the cell extract of a shake flask experiment using the Qiagen Ni-NTA protocol. This F was found to be active against RSV in an ELISA assay.

1.2 Solubility Assay

A 0.975 mL volume of culture was centrifuged in a microfuge for 5 minutes at 14,000 RPM. The supernatant liquid was decanted and the cells were resuspended in lysis buffer up to the starting volume, wherein Lysis buffer comprised the following components: Tris HCl, 50 mM, pH 7.5 final; NaCl, 200 mM; glycerol, 5%v/v; EDTA, 20 mM; Triton X-100, 5%v/v; and, added last, DTT, 1 mM. Screw-cap microfuge tubes (2 mL) were filled about 3/4 full with 0.1 mm glass beads and topped off with cell suspension. The tubes were given a quick shake to mix the beads and removed air bubbles, and further filled to the top with cell suspension. The tubes were capped, sealed, and inserted into a BioSpec mini bead-beater for 60 seconds at 5000 rpm. The samples were kept on ice between beatings and beat 3 to 5 times until about 90%of the cells were lysed. Cell lysis was observed by microscopic observation. A volume of 0.025 mL of the lysed cell preparation was pipetted from each tube, minus beads, into new microfuge tubes and centrifuged for 5 minutes. The supernatant fraction was carefully transferred to another tube with 0.075 mL LSB, and 0.100 mL LSB was added to the pellet fraction. The supernatant and pellet fractions were re-suspended with a Vortex stirrer, the tubes were capped, placed in a boiling water bath for five minutes, and 0.005 mL to 0.010 mL aliquots of the fractions SDS PAGE were analyzed. Assessment of expressed SARS-COV F protein solubility in Pseudomonas, using either a French-Press or a BioSpec Mini Bead-Beater produced equivalent results.

The solubility of SARS-COV F in cells was tested bacteria and indicated that most, if not all, of the SARS-COVF remained in soluble form. To do these solubility-tests, viable un-amended bacteria cells were broken in a French Press (or mini-bead beater) , and centrifuged to separate cell debris and any inclusion bodies from soluble proteins. SDS gels of these two fractions indicated that SARS-COV F was retained in the soluble portion.

SDS-PAGE analysis of French-pressed E. coli cultures containing SARS-COV F was conducted. E. coli cells were ruptured in a French Press and centrifuged at 16000 g for five minutes. Supernatant samples showed a single, major band (about 75 kDa) of soluble SARS-COV F. Pelleted samples showed a major band (about 75 kDa) of SARS-COV F together with small amounts of contaminating soluble SARS-COV F. The contamination appears to be due to spillover from the supernatant fraction and unlysed cells.

Both the amount and activity of SARS-COV F in E. coli cells were high. As illustrated in the examples, bacteria is a good biofactory, capable of producing up to 40%or more of total cell protein as recombinant protein, such as SARS-COV F and the cells produced active protein.

Cell culture medium was harvested, loaded into the Ni-NTA column and washed with washing buffer (50 mM Na₂HPO₄, 300 nM NaCl, 20 mM imidazole, pH 8.0) , and eluted with elution buffer washing buffer containing 250 nM imidazole) , and purified from the soluble fraction by denaturing Ni-NTA chromatography.

The purified fusion protein was incubated with (Factor Xa +TEV 1: 1) protease (Novagen) at room temperature in a buffer containing 20 mM Tris, pH 8.0, 0.2 M NaCl and 5 mM CaCl₂. One unit of Factor Xa was used for 75–100 μg of target protein. The reaction was allowed to proceed for 36–48 h for maximal cleavage (90–95%) of the fusion protein. The fractions were concentrated using Amicon Ultra concentrator (Millipore) with a 10 kDa cut-off filter. Protein concentration was determined by measuring absorbance at 280 nm using multi-mode microplate reader (BioTek Synergy 2) . Protein purity and integrity were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by visualization of the protein using PAGE Blue stain (Invitrogen) . PAGE Blue staining results of purified F protein, F monomers, Trimers F were shown in Fig. 1. Negative stain electron microscopy of Rag was shown in Fig. 2, indicating that the RAg was highly homogenous.

Example 2 Preparation of Nanoparticles

For preparation of nanoparticles, PLGA (mol wt 40-75 kDa) , polyvinyl alcohol (mol wt. 30-70 kDa) (Sigma-Aldrich) , Dicholoro methane (Acros Organics) , and BCA (bicinchoninic acid) protein assay kit (Pierce) were used.

1. Materials and Methods

1.1 Production of WCL (Whole Cell Lysate)

Four selected non-pathogenic Mycobacterium species can be grown in liquid medium as recommended by ATCC (American Type Culture Collection) . BCG (Bacillus Calmette-Guerin) was purchased from National Institute for Food and Drug Control and WCL can be prepared. Live bacteria can be harvested and washed twice using PBS (pH 7.4) and suspended (2 g/ml) in PBS containing 8 mM EDTA, proteinase inhibitors, DNase and RNase. Cells can be disrupted by using the Bead Beater until approximately 90%breakage was obtained (monitored by acid fast staining) , centrifuged at 3,000×g to pellet unbroken cells and insoluble cell wall components, and the supernatant (WCL) can be harvested. The protein content and endotoxin levels in the WCL can be quantified using the kits, and the aliquots will be stored at -70℃. The mycobacterial WCL preparation contains water-soluble proteins, lipids, and carbohydrates.

1.2 Preparation of PLGA Nanoparticles

Nanoparticles were prepared by a standard double emulsion solvent evaporation method. Briefly, 15%of PLGA (1500 mg) was dissolved in 5 ml of dichloromethane and 10 mg F protein was added. The mixture was homogenized for 90 seconds using a homogenizer at 6000 rpm. The homogenized mixture was added to 120 ml of aqueous solution of polyvinyl alcohol (10%PVA) , and homogenized for 5 min. Finally, the preparation was stirred overnight at room temperature (RT) to allow solvent evaporation. The nanoparticles were washed in distilled water for three times and the wet nanoparticles were freeze-dried and stored at 4 ℃.

1.3 Preparation of Vaccine Antigens and PLGA Nanoparticle-Based Vaccine Formulations

RSV F antigens as described previously used in the Example 1, PLGA NPs entrapped with RAg (NP-RAg) or M. tb (Mycobacterium tuberculosis) WCL (NP-M tb WCL) were prepared by double emulsion method (w/o/w) .

1.4 Determination of Size, Morphology, and Protein Entrapment Efficiency of Nanoparticles

The size and morphology of nanoparticle was detected using scanning electron microscopy. Freeze-dried nanoparticles were mounted on an adhesive stub coated with gold platinum under vacuum using an ion coater. The coated specimen was examined under the microscope at 10 KV. The amount of entrapped r F in the nanoparticles was determined.

1.5 Characterization of NP-RAg

Entrapped protein in NPs was estimated. Morphology of the Nano-RAg was visualized using the Philips XL30-FEG scanning electron microscope (SEM) at 20 kV at 30,000× magnification. Size distribution of the sham or RAg entrapped NPs was measured using NICOMP 370 particle sizer (Particle Sizing Systems, CA) . The zeta potential of the NPs was determined by ZetaPALS (Brookhaven Instruments Corp., NY) .

1.6 Determination of In Vitro Protein Release from Nanoparticle Entrapped recombinant F (r F) (NP-RAg)

The assay was performed, 100 mg NP-RAg was suspended in two ml PBS and the supernatant was collected immediately to estimate the burst release. The pellet of NP-RAg was resuspended repeatedly with two ml PBS and the supernatants were collected at 1, 5, 10, 15, 20, 25, and 30 days and stored at -20 ℃. On day 30, undegraded NPs were lysed to recover the protein, and all the samples were estimated for protein concentration by BCA method.

1.7 Immunogenicity of the encapsulated and released F proteins

ELISA plates were coated with 5 μg/mL (2.5 μg/mL each) of the recombinant F proteins (BRSV-F) , or with 100μL/well of RSV virus stock (～10⁴ TCID₅₀) grown in Hep4 cells. Recombinant RSV F proteins were encapsulated in PLGA particles and released as described. The released proteins were also coated onto ELISA plates at ～5μg/mL (BRSV-F NP) . Sera from RSV-immune rats were diluted 1: 1000 and added to the plates. The binding of rats IgG to the virus or recombinant proteins was measured by absorbance.

PBMC were labeled with Cell Trace Violet and stimulated for 6 days with 5 μg/mL of the recombinant RSV F proteins; 5μg/mL recombinant RSV F proteins that were encapsulated and released from the PLGA particles; or 0.01 MOI of RSV strain Long. Pokeweed Mitogen was used at a concentration of 1μg/mL as a positive control. Mock stimulated samples (negative control wells) were cultured with cRPMI and were used to correct for background proliferation. After 6 days, antigen-specific CD4 T cell proliferation was assessed by flow cytometry. Results are pooled from 2–3 independent experiments for a total of n=8–10 animals. Data represent mean±SEM. *p<0.05 p**<0.01 NS: not significant.

1.8 In Vitro Uptake of Nano-RAg by PAM Cells

Nano-RAg vaccine containing 4 μg of RSV RAg were suspended in one ml of RPMI and incubated with PAM (ATCC) for 0, 5, 20, 30 min, 3, 12, and 24 hr. RAg (4μg) , sham NPs, and HEp-2 cells infected for 24 hr with RSV strain Long (ATCC) were included as controls. Cells were washed and fixed with acetone and incubated with RSV F mAb followed by anti-mouse IgG (H+L) Alexa-488 (Life Technologies, Grand Island, N. Y. ) , and observed under the inverted fluorescent microscope.

1.9 In vitro analysis of nanoparticle immunogenicity in cotton rats APC

Monocyte-derived dendritic cells (moDC) were prepared from adult rats. The moDC were generated. Recombinant rats IL-4 and recombinant rats GM-CSF were purchased from (JDK, Beijing) . Rat moDC were seeded at 5×10⁵ cells per well in a 24-well plate and stimulated with 10 μg/mL ‘empty’ or RSV-F-loaded PLGA particles. Mock cultures were treated with media only. After 18 hours, RNA was isolated from the cells and analyzed by qPCR for expression of IL-8 and IL-12p40. For qPCR analysis, results were normalized to the housekeeping gene RPS-9, and expressed relative to unstimulated control samples. After 48 hours, cell culture supernatants were collected and analyzed by ELISAs for concentrations of IFNγ, IL-6, IL-1β and TNFα. Alveolar macrophages were isolated from the BAL fluid of healthy rats and were seeded at a concentration of 5×10⁵ cells per well in 24-well plates. The macrophages were stimulated with empty PLGA particles or with PLGA nanoparticles that were loaded with RSV-F from the RSV F protein. Mock cultures were treated with media only. After 48 hours, cell culture supernatants were analyzed by multiplex immunoassay. Results were pooled from two independent experiments for a total of n=8–10 animals. Data represent mean±SEM *p<0.05 **p<0.01 compared to mock stimulated cultures.

2.Results

2.1 Size, Morphology, Protein Entrapment Efficiency and Characterization of Nanoparticles

NP-RAg vaccine was rapidly endocytosed by cotton rat alveolar macrophages (MAMs) and sustained release of entrapped NP-RAg over several weeks. Potency of NP mediated delivery of drugs or vaccines depend on their loading capacity and size. The entrapment efficiency of RAg (rRSV F) and M. tb WCL in NPs was about 50-60%. Sham or entrapped NP particles were circular with smooth surface. Dynamic light scattering (DLS) of NPs measured their diameter based distribution, and the mean diameter±SD of sham, NP-RAg, and 151 NP-M. tb WCL were 490±45, 510±44, and 670±76 nm, respectively. Further, 85%of sham NPs, 92%of NP-M. tb WCL, and 78%of NP-RAg were in the size range of 400-700 nm. Interestingly, there was no difference among all three NPs with respect to surface electrostatic potential (-26 mV) as measured by Zeta potential, indicating that differential surface charge of entrapped proteins did not influence the net electrostatic potential of finally formed NPs.

The morphology of RAgs entrapped PLGA nanoparticles was shown in Fig. 3. Scanning electronic photomicrograph of PLGA nanoparticles prepared by a standard multiple emulsion method. The size of the nanoparticles appears to be variable ranging 400–700 nm. The encapsulation efficiency of F protein in then nanoparticles was 20%.

2.2 In Vitro Protein Release Profile of Nanoparticle Entrapped recombinant F (r F) (NP-RAg)

The surface associated protein in nanoparticles was equivalent to the amount of protein released at time zero (i.e., immediately after reconstitution in PBS) , called as burst release and it was 9.5%in NP-RAg vaccine. The release during first 24 hr after reconstitution was 30.5%, and after 30 days 61%of the entrapped protein was released (as shown in Fig. 4) . The remaining 39%of viral Ag was recovered in the un-lysed NPs. Even one year old NP-RAg stored at -20 ℃, NP-RAg had 13.6%burst release, and 76%of released antigens by 30 days, indicating that PLGA NPs retain the entrapped vaccine beyond one year and allow its sustained release over a period of several weeks under normal physiological conditions. Thus, the results indicated that as expected PLGA nanoparticles could efficiently retain and permit sustained release of entrapped RSV Ag over a long period of time.

2.3 Immunogenicity of the encapsulated and released F proteins

Immunogenicity of recombinant RSV-F was preserved following release from PLGA nanoparticles. The recombinant RSV F was immunogenic and stable following encapsulation and release from the PLGA nanoparticles. As shown in Table 1-2, CD4 T cells from RSV-immune cows underwent clonal expansion in recall response to RSV, recombinant RSV-F and the released RSV-F/G proteins. We observed no significant differences in the response to the encapsulated and released proteins compared to the unencapsulated recombinant proteins.

Table1

Table. 2

2.4 In Vitro Uptake of NP-RAg Vaccine by PAM Cells

RSV is host-specific, infects cotton rats and the virus infects pulmonary alveolar macrophages (PAM) . Treatment of PAM cells with the vaccine preparations revealed very little uptake of unentrapped RAg over 24 hr. In contrast, a rapid uptake of NP-RAg as early as 5 min and reached the peak by 30 min, followed by a gradual reduction in the fluorescence signals after 3 hr post-treatment; possibly due to degradation of Ags. Control sham nanoparticles treated and untreated PAM cells did not show any fluorescence signals, while the RSV infected cells had virus specific green signals.

2.5 Inflammatory cytokine production measured by multiplex immunoassay

As shown in Fig. 5A-Fig. 6D, alveolar macrophages were stimulated with 10 μg/mL PLGA nanoparticles that were ‘empty’ . Inflammatory cytokine production was measured by multiplex immunoassay. Increased inflammatory cytokine expression by nanovaccine-stimulated alveolar macrophages compared to untreated controls were observed and observed no difference in the response to ‘empty’ or RSV-F loaded nanoparticles. Together with our results confirmed that the PLGA nanoparticles were able to activate rat APC and demonstrated that this response was largely independent of the antigen payload, as ‘empty’ particles and particles loaded with recombinant F proteins elicit similar inflammatory responses.

Example 3. Nanoparticle-Based Adjuvanted RSV F Vaccine Elicits Superior Protective Immunity in cotton rats

1.Materials and Methods

1.1 Safety and Ethics

All experiments and procedures involving cotton rats were approved under protocol LVD29E by the National Institute of Viral Diseases Prevent and Control Animal Care and Use Committee according to standards set forth in the cCDC guidelines, Animal Welfare Act. Euthanasia was carried out using carbon dioxide inhalation in accordance with the China Veterinary Medical Association Guidelines for Euthanasia of Animals (Experiments with RSV were carried out under biosafety level-2) . Animals Five-to 6-wk-old female cotton rats were purchased from SPF Biotechnology (Beijing) , and were housed in a BSL-2 facility at Viral Diseases Prevention and Control.

1.2 Cells, Virus and Antigens

HEp-2 cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO-BRL, ) with 10%fetal rat serum (FBS, GIBCO-BRL) , 2mM glutamine, penicillin and streptomycin (GIBCO-BRL) at 37℃ with 5%CO₂.

RSV (strain Long) was kindly provided by Dr Xu Wenbo (National Institute of Viral Diseases Prevention and Control, Beijing) and propagated in HEp-2 cells.

1.3 Nanoparticle Vaccine Studies

Peripheral blood and sera were collected from eight female adult cotton rats housed at Viral Diseases Prevention and Control. Animals from this herd have no RSV-specific antibodies and RSV-specific CD4 T cells.

Twenty-four, cotton were enrolled at 5–6 weeks of age and were randomly assigned to four treatment groups (n=6 animals/group) : the various groups used for the study are presented in Table 3, RAg+WCL, PLGA-RAg, PLGA-RAg+WCL; PLGA+WCL intranasal vaccination and challenged with RSV strain Long. Each rats received ～10 μg total of the recombinant RSV-F proteins IN. Vaccines were administered in a volume of 1mL with 0.3 mL injected into each nostril. Nasal fluid samples were collected at weekly intervals following vaccination, and on days 2, 4 and 6 post RSV challenge. Commercial, polyurethane sponges were cut into 1–2 inchs squares and autoclaved. Sponges were dampened with 1mL of sterile saline and then a single square was inserted into the rat’s nostril for 5–10 minutes. The sponges were then removed, placed in a tube and an additional 1mL of sterile saline was added. Liquid was recovered from each sponge by squeezing in the barrel of a 5mL syringe. The resulting nasal fluid was then aliquoted and frozen at -80 ℃ for later use.

Table 3. Groups for animal experiment.

I.N. =intranasal, MP=nanoparticle. N/A=not applicable.

Cotton rat (n = 5, per group) were dosed with PLGA-RSV-F nanoparticle with or without adjuvant. The control group of mice was administered PLGA plus WCL and served as the negative control, while the recombinant RSV F plus WCL administered group was selected as a positive control.

1.4 Challenge Virus

RSV long strain was propagated in confluent HEp-2 cell and 10⁵ TCID50 was administered IN to cotton rats that were lightly sedated with isoflurane.

RSV infected Hep-2 cells were cultured for 3 days, harvested and centrifuged for 10min at 2000 rpm in a table-top centrifuge at 4 ℃. RSV within supernatants wascollected. Post-fusion (F) stabilized F and pre-fusion stabilized F proteins and pre-fusion specific 5C4 mAb were generously provided by Dr XuWenbo (National Institute of Viral Diseases Prevention and Control, Beijing) . Palivizumab mAb was kindly provided by Dr XuWenbo (National Institute of Viral DiseasesPrevention and Control, Beijing) . D25 mAb was purchased from Creative Biolabs (Shirley, NY, USA) .

1.5 Necropsy and Pathological Evaluation

Rats were euthanized on day 7 post-infection (p.i. ) by barbiturate overdose. Pathological evaluation was performed. Bronchoalveolar lavage fluid (BAL) was collected by introducing 500 mL of sterile, ice-cold PBS through the trachea. Samples of affected and unaffected lungs were collected from multiple sites for histopathological evaluation. Histopathology Lung tissues collected from cotton rats at 5 days after RSV challenge were fixed with 10%neutral buffered-formalin. Lung tissue histology was performed by staining with hematoxylin and eosin (H&E) , periodic acid–Schiff (PAS) , and Congo red (C&R) and analyzed under light microscopy. The tissue slides were examined for lymphocytes and eosinophils in peribronchiolar, perivascular, interstitial, and alveolar spaces. At least 10 sections per lung tissue from individual rats were obtained and blind scoring was performed for histopathology analysis.

Inflammation and focal aggregates of infiltrating cells in the airways of the lung were blindly examined and measured using a severity score system defined as 0 (normal) , 1 (mild inflammation, 60%lung affected with tissue necrosis or damage) . The mucin expression of goblet cell hyperplasia was identified in 50 randomly selected lung airways in the PAS stained slides. Eosinophils were counted per viewing PAS-positive areas within the airway epithelium (400× magnification) and annotated using the magnetic lasso tool of Adobe Photoshop CS5.1 software.

1.6 Viral Load

An method for determining viral load is quantitative real-time PCR (qRT-PCR) . Viral load can be determined by qRT-PCR using oligonucleotide primers specific for the RSV-F gene. Briefly, RNA is isolated from 140 μl of clarified lung homogenate, or from a known number of plaque forming units (PFU) of RSV (determined by plaque assay, and diluted in lung homogenate from uninfected animals) , using the RNeasy kit (Qiagen) with a final elution volume of 100μl H₂O. cDNA synthesis and PCR was performed in a single tube using the SuperScript III Platinum One-Step Quantitative RT-PCR kit (Invitrogen) with 5μL of eluted RNA, 10μM of each primer, and 50μM of the probe (primers and probes from Integrated DNA Technologies) . Forward primer: TTGGATCTGCAATCGCCA (SEQ ID NO: 10) . Reverse primer: CTTTTGATCTTGTTCACTTCTCCTTCT (SEQ ID NO: 11) . Probe: 5'-carboxyfluorescein (FAM) -TGGCACTGCTGTATCTAAGGTCCTGCACT-tetramethylcarboxyrhodamine (TAMRA) -3' (SEQ ID NO: 12) . Amplification and detection was performed with an ABI Prism 7900HT or 7500 (Applied Biosystems) . A threshold cycle value (Ct) is defined for each sample as the cycle number at which the fluorescent signal first becomes detectable above a set threshold. PFU equivalents for each sample is then determined based on a standard curve of Ct verses the logarithm of defined copy number of viral RNA.

1.7 RSV-specific IgA in the nasal and BAL fluid of PLGA-RAg+WCL nanoparticle vaccine cotton rats

ELISAs were performed according to kit manufacturer’s instructions. Indirect ELISAs were used to quantify IgA in the nasal and BAL fluid. Indirect ELISAs were also used to determine the immunogenicity of the RSV F proteins prior to encapsulation and following release from the PLGA nanoparticles. For the IgA quantification, ELISA plates were coated overnight at 4 ℃ with 3 μg/mL F protein, or with 100 μl/well of RSV stock (～10⁵ TCID50) . For the immunogenicity studies, the ELISA plates were coated overnight at 4 ℃ with 5μg/mL total of the F protein (2.5 μg/mL of each) , with ～5 μg/mL of the RSV F proteins that had been encapsulated and released from the PLGA nanoparticles, or with 100 μl/well of RSV stock (～10⁵ TCID50) . Negative control wells were coated with 100 μl/well cell culture media prepared from uninfected HEp-2 cells. Nasal fluid samples were diluted 1: 2 and treated with 10mM dithiothreitol (DTT) for 1 hour at 37 ℃ prior to performing the ELISAs. BAL samples were diluted 1: 2 but were not treated with DTT. Serum samples were diluted 1: 1000. All samples were plated in duplicates, incubated for 2 hours at room temperature and then washed. Sheep anti-rat IgA-HRP (Bethyl Laboratories) was used at 0.5 μg/mL. Sheep anti-rat IgG-HRP (Bethyl Laboratories) was used at 0.5 μg/mL for the immunogenicity experiments. Plates were developed using Pierce 1-Step Ultra TMP Substrate (Termo Scientifc Pierce) . The reaction was stopped with the addition of 0.2M H₂SO₄ and plates were read at an optical density of 450nm and 540nm using an automated plate reader.

1.8 Cytokine secretion in supernatants from rats alveolar macrophages

A multiplex immunoassay (Invitrogen) was used to quantify cytokine secretion in supernatants from rats alveolar macrophages. IL-17A, IFNγ, IL-6, IL-1β and TNFα Set ELISA Development kits were purchased from King fisher Biotech, Inc. The bovine IL-4 ELISA kit was purchased from Termo Fisher Scientific ELISAs were performed according to kit manufacturer’s instructions. Indirect ELISAs were used to quantify IgA in the nasal and BAL fluid. Indirect ELISAs were also used to determine the immunogenicity of the BRSV F and G proteins prior to encapsulation, and following release from the CPTEG: CPH nanoparticles. For the IgA quantification, ELISA plates were coated overnight at 4 ℃ with 3μg/mL F or G protein, or with 100 μl/well of BRSV stock (～10⁴ TCID50) . For the immunogenicity studies, the ELISA plates were coated overnight at 4 ℃ with 5μg/mL total of the F and G protein (2.5 μg/mL of each) , with ～5μg/mL of the BRSV F and G proteins that had been encapsulated and released from the CPTEG: CPH nanoparticles, or with 100 μl/well of BRSV stock (～10⁴ TCID50) . Negative control wells were coated with 100 μl/well cell culture media prepared from uninfected BT. Nasal fluid samples were diluted 1: 2 and treated with 10mM dithiothreitol (DTT) for 1 hour at 37 ℃ prior to performing the ELISAs. BAL samples were diluted 1: 2 but were not treated with DTT. Serum samples were diluted 1: 1000. All samples were plated in duplicates, incubated for 2 hours at room temperature and then washed. Sheep anti-bovine IgA-HRP (Bethyl Laboratories) was used at 0.5 μg/mL. Sheep anti-bovine IgG-HRP (Bethyl Laboratories) was used at 0.5μg/mL for the immunogenicity experiments. Plates were developed using Pierce 1-Step Ultra TMP Substrate (TermoScientifc Pierce) . The reaction was stopped with the addition of 0.2M H₂SO₄ and plates were read at an optical density of 450nm and 540nm using an automated plate reader

1.9 RSV F-Specific ELISA

Individual serum samples were assayed for the presence of RSV F-specific IgG by enzyme-linked immunosorbent assay (ELISA) . ELISA plates (MaxiSorp 96-well, Nunc) were coated overnight at 4 ℃ with 1 μg/ml purified RSV F in PBS. After washing (PBS with 0.1%Tween-20) , plates were blocked with Superblock Blocking Buffer in PBS (Thermo Scientific) for at least 1.5 hours at 37 ℃. The plates were then washed, serial dilutions of serum in assay diluent (PBS with 0.1%Tween-20 and 5%goat serum) from experimental or control cotton rats were added, and plates were incubated for 2 hours at 37 ℃. After washing, plates were incubated with horse radish peroxidase (HRP) -conjugated chicken anti-cotton rat IgG (Immunology Consultants Laboratory, Inc, diluted 1: 5,000 in assay diluent) for 1 hour at 37 ℃. Finally, plates were washed and 100 μl of TMB peroxidase substrate solution (Kirkegaard &Perry Laboratories, Inc) was added to each well. Reactions were stopped by addition of 100μl of 1M H₃PO₄, and absorbance was read at 450 nm using a plate reader. For each serum sample, a plot of optical density (OD) versus logarithm of the reciprocal serum dilution was generated by nonlinear regression (GraphPad Prism) . Titers were defined as the reciprocal serum dilution at an OD of approximately 0.5 (normalized to a standard, pooled sera from RSV-infected cotton rats with a defined titer of 1: 2500, that was included on every plate) .

1.10 Micro Neutralization Assay

Serum samples were tested for the presence of neutralizing antibodies by a plaque reduction neutralization test (PRNT) . Two-fold serial dilutions of HI-serum (in PBS with 5%HI-FBS) were added to an equal volume of RSV Long previously titered to give approximately 115 PFU/25μl. Serum/virus mixtures were incubated for 2 hours at 37 ℃ and 5%CO₂, to allow virus neutralization to occur, and then 25 μl of this mixture (containing approximately 115 PFU) was inoculated on duplicate wells of HEp-2 cells in 96 well plates. After 2 hours at 37 ℃ and 5%CO₂, the cells were over-laysed with 0.75%Methyl Cellulose/EMEM 5%HI-FBS and incubated for 42 hours. The number of infectious virus particles was determined by detection of syncytia formation by immunostaining followed by automated counting. The neutralization titer is defined as the reciprocal of the serum dilution producing at least a 60%reduction in number of synctia per well, relative to controls (no serum) .

1.11 Statistical Analysis

All statistical analyses were performed using Prism software (GraphPad Software, Inc., San Diego, CA) . Data were evaluated using either one-way ANOVA with Tukey’s post hoc test or 2-way ANOVA with Dunnett’s post hoc test for comparison between more than two groups. A value of p <0.05 was considered significant.

2. Results

2.1 In vivo immunogenicity and efficacy of the RSV-F nanoparticles

To determine the efficacy of the RSV-F PLGA nanoparticles vaccine in rats, animals were vaccinated IN with 10 μg RSV-F plus WCL, 10 μg ‘empty’ PLGA nanoparticles plus WCL, 10 μg RSV-F -loaded nanoparticles plus WCL, and 10 μg ‘empty’ PLGA nanoparticles plus WCL, respectively. Four weeks later, rats were challenged via intranasal inoculation with ～10⁵ RSV strain Long. Rats were monitored daily for clinical signs and rectal temperatures. Several animals in the vaccinated group demonstrated elevated temperatures (40–41 ℃) for 1–2 days during infection; however, the fevers were not prolonged and no significant differences in body temperature were observed between vaccinated groups. Mild clinical signs were observed in the RSV challenged animals, including increased respiration rates and increased expiratory effort. Clinical signs of experimental RSV infection were apparent starting on days 4–6 after infection and were recorded in at least some animals from each group. Mild clinical signs were observed in 3/6 rats in the R -Ag+WCL vaccinated group, 4/6 cotton rats in the ‘empty’ nanoparticles+WCL administered groups and 2/6 rats in the RSV-F nanoparticle +WCL administered group. However, due to variability between animals and disease kinetics, the clinical scores did not differ significantly between vaccinated groups (presented in Table 4) . No clinical signs were observed in the uninfected control animals at any time during the experiment.

Table4 Aggregate gross pathology results from all groups and all animals

2.2 Pathological Evaluation

Animals were euthanized on day 7 p.i. The lesions were bilateral and most frequently observed in the cranioventral lung lobes, consisting of multifocal to coalescing areas of firm, pneumonic consolidation (Fig. 7) . Unvaccinated control animals developed large, diffuse gross lesions, affecting as much as 40–50%of the lung, while rats receiving the RSV nanoparticle vaccine (PLGA-RAg+WCL) demonstrated fewer lesions and a significant reduction in the area of lung affected (Fig. 7) . Representative micrographs from an uninfected control rats (i) , an unvaccinated positive control rats (ii) , an ‘empty’ nanoparticle-administered rats (iii) and a RSV-F/vaccinated rats (iv) are shown in Fig. 7. Cumulative histopathology scores were depicted in Table 5. Pulmonary lesions were most pronounced in the unvaccinated control animals and included thickened alveolar septa with infiltrates of macrophages, lymphocytes and occasional neutrophils; and bronchioles filled with neutrophils, sloughed epithelial cells and necrotic cell debris. Six of the six RSV-F nanoparticle (PLGA-RAg+WCL) -vaccinated rats exhibited reduced histological lesions compared to the PLGA+WCL vaccinated rats. Four of six RSV-F nanoparticle (PLGA-RAg) vaccinated rats compared to the PLGA+WCL vaccinated rats. Two of the six RSV-F vaccinated rats compared to the PLGA+WCL vaccinated rats, exhibited reduced histological lesions with only mild peribronchiolar lymphocytic infiltration and minor accumulation of alveolar exudates. No evidence that the RSV-F nanoparticle promoted the development of enhanced or exacerbated disease was observed.

Table 5 Aggregate microscopic pathology

2.3 Viral Load

Reduced viral burden and reduced virus shedding in RSV-F nanoparticle-administered rats. Nasal swabs and lung tissues were assessed for virus isolation. RSV was isolated from 5/6 PLGA+WCL vaccinated cotton rats on day 3 p. i and 6/6 animals on day 6 p.i. Virus was isolated from the lungs of all 6 PLGA+WCL vaccinated controls at necropsy. In contrast, RSV was isolated from 0/6 PLGA-RAg+WCL vaccinated cotton rats on day 3, and 1/6 on day 6 p.i. Virus was isolated from the lungs of this same animal on day 7 p.i. RSV was isolated from the nasal swabs of 4/6 cotton rats in the ‘empty’ PLGA+WCL administered group on days 3 and 6 after infection, and from the lung tissues of 3/6 cotton rats on day 7 p.i. Virus from the nasal swabs collected from any of the animals on day 0 (prior to RSV challenge) was not isolated , nor RSV from the nasal swabs or lung tissues of the uninfected control cotton rats.

qPCR for the RSV NS2 gene on lung tissues collected on day 7 p.i. was performed.

RSV-F nanoparticle vaccine (PLGA-RAg+WCL) i.n. administered cotton rats demonstrated significantly reduced quantities of viral RNA compared to their PLGA+WCL cohorts (Fig. 8) .

Fig. 8 showed the reduced viral burden in the lungs of BRSV-F nano vaccine-administered rats. Samples were collected from 2–3 representative lesion and non-lesion sites of the lungs on day 7 post-challenged and preserved in RNA later. The RNA was extracted using Trizol reagent. The RNA from multiple sites was then pooled and was analyzed by qPCR for the RSV NS2 gene. Viral NS2 copy numbers were calculated using standard curves and normalized to the housekeeping gene, S9, to correct for differences in input material. Results represent the mean NS2 copy number of each rat, with n=6 animals/group. The graph depicts mean±SEM of each group. *p<0.05 compared to PLGA+WCL control rats.

2.4 RSV-specific IgA in the nasal and BAL fluid of PLGA-RAg+WCL nanoparticle vaccine cotton rats.

Nasal fluid samples were assessed for RSV-F-specific IgA following intranasal vaccination and challenge. Significant changes in RSV-specific IgA on day 28 after vaccination was observed. By day 6 after infection, PLGA-RAg+WCL vaccinated cotton rats demonstrated a significant increase in virus-specific IgA compared to the PLGA+WCL vaccinated cotton rats or animals receiving the R-Ag+WCL (Fig. 9A) . Increased RSV-F specific IgA in the BAL fluid from PLGA-RAg+WCL, PLGA-RAg administered cotton rats, by day 7 after infection (Fig. 9B) was also observed. Virus-neutralization assays using nasal fluid to determine if neutralizing antibody responses were generated in the respiratory tract. On day 28-post vaccination (day 0 prior to challenge) , low titers of neutralizing antibody in the nasal fluid of all vaccinated animals, with no significant differences observed between treatment groups (Table 6) . By day 6 after infection, an increase in neutralizing antibody titers in the nasal fluid of all RSV-challenged groups was observed, band noted significantly higher titers (p≤0.01) in the nasal fluid of PLGA+WCL nanoparticle vaccine -administered animals compared with the PLGA+WCL vaccinated control animals. Importantly, these results indicate that a single dose of the PLGA+WCL nanoparticle promotes significant production of neutralizing, RSV-specific antibodies in the respiratory tract of cotton rats, given the importance of developing an RSV nanoparticle vaccine that is efficacious.

Table 6 Virus neutralization titers measured in the nasal fluid

Virus neutralization titers measured in the nasal fluid on day 28 (day 0 before challenge) and day 6 post-challenge. Data are presented as the mean (range) titer per group. **p<0.01 compared to the PLGA+WCL group.

Cotton rats in all groups demonstrated BRSV-specific IgG in the serum, with neutralizing antibody titers ranging from 16–256 on the day of vaccination; 16–128 on day 28 post-vaccination (day 0, prior to challenge) ; and 8–128 on day 6 after challenge. Significant differences were presented between treatment groups. Significant differences changes in serum neutralizing titers related to RSV-F/G nanovaccine-administration or RSV challenge.

RSV-specific cellular responses in the lungs and peripheral blood of RSV-F nanovaccine administered rats. Prior to challenge, we detected significant RSV-F protein specific CD4 or CD8 T cell proliferation in PBMCs from any groups. By day 6 p.i., we measured significant antigen-specific proliferation by CD4 T cells from RSV-F nanovaccine-administered rats in response to live virus a (Fig. 10A) . PBMCs from the BRSV-F nanovaccine-administrated animals also secreted IFNγ and IL-17A in response to whole virus (Fig. 10B-10C) . Levels of IL-4 were below the limit of detection for all groups (data not shown) . Mononuclear cells were isolated from the BAL on day 7 p.i. and stimulated with whole virus, F protein. We measured a significant increase in the concentration of IFNγ and IL-17 in the BAL cell culture supernatants from the BRSV-G nanovaccine-administered rats, but not control rats (PLGA+WCL) , in response to protein or whole virus stimulation.

This invention achieves advantageous effect

RSV infection has a devastating, worldwide impact on human health and despite significant efforts, no approved vaccine currently exists for use against the disease in humans. Herein, a single, i.n. administration of the RSV-F induced mucosal, anti-viral immunity and protected most animals from virulent RSV challenge. Rats receiving the BRSV-F nanovaccine mounted cellular and humoral immune responses in the upper and lower respiratory tract (Figs 9 and 10 and Table 6);exhibited a reduced viral burden in the lungs on day 7 post challenge (Fig. 8) ; and developed significantly fewer gross and microscopic lesions in the lungs compared to unvaccinated control animals (Fig. 7) . Our PLGA nanovaccine platform, which is based upon PLGA copolymers, offers a number of advantages over other polymeric nanoparticle systems, including: inherent adjuvant properties the tendency of the particles to surface erode, thus stabilizing the encapsulated proteins and maintaining the structural and biological features of the antigens for longer periods of time; the ability to provide sustained and tunable antigen release kinetics; and the ability to degrade at a neutral pH, into non-toxic and non-mutagenic carboxylic acids. Herein that the in vitro antigenicity of the RSV F proteins was preserved following encapsulation and release from the PLGA particles (Fig. 4) . We observed no significant changes in the capacity of RSV-specific polyclonal antibodies or RSV-specific CD4 T cells to recognize the recombinant proteins following their release from the nanoparticles, which is critical for vaccine efficacy. The length of antigen exposure is a vital factor dictating the establishment of long-term immunity. The PLGA nanoparticles used here provided sustained antigen release of the RSV F protein, with only ～40%of the recombinant protein released by 30 days in vitro. The RSV-F nanovaccine should be stable and immunogenic, our in vivo vaccine experiments demonstrated that a single i.n. vaccination, containing a suboptimal dose of antigen, was suffcient to protect a majority of cotton rats from severe RSV infection. Both neutralizing IgG and IgA are thought to have a role in protection from RSV.

Our unvaccinated control animals (PLGA+WCL) developed RSV disease, including transient fevers, clinical signs, severe pathology in the lungs and signifficant viral shedding. Adults who have been repeatedly infected with RSV develop sustained high levels of IgA in nasal secretions, which has been shown to prevent virus replication in the upper airways, regardless of serum Ig levels, Mucosal IgA also plays an important role in reducing the occurrence and severity of RSV infection in infants and children Our results reinforce this conclusion, demonstrating that RSV-F vaccinated rats mounted a significant IgA response in the nasal cavity and BAL that increased rapidly in the days following virulent RSV challenge. However, in adults, IgA responses can wane and the IgG response may be more important in long-term protection. We observed a significant rise in neutralizing antibody titers in the nasal secretions of the BRSV-F nanovaccine administered rats (Table 6) . Based upon the results of our ELISA analysis, this response is presumably mediated by neutralizing IgA.

RSV infection causes inflammation in the upper respiratory tract, thus the increase in neutralizing antibody titers could also potentially be attributed to increased levels of serum antibodies leaking into the nasal secretions of the ‘empty’ nanovaccine administered rats.

The RSV F protein is a type I viral fusion protein that is synthesized as a precursor that is proteolytically cleaved by furin into disulfde-linked fragments. It is highly conserved between virus strains, as well as between HRSV and BRSV, demonstrating approximately 80%homology. The F protein exists in two forms on the virion surface: a metastable pre-fusion form and a stable post-fusion trimer. The post-fusion form of the F protein contains two major neutralizing epitopes, antigenic sites II and IV7. High-affinity, site-II directed neutralizing antibodies are protective in cotton rats. When considering the design of an F protein based subunit vaccine, protein stability is of paramount concern, and the post-fusion F is advantageous due to its highly stable nature. The pre-fusion F protein contains antigenic siteand recent evidence suggests that this epitope is the primary target for neutralizing antibodies in humans. Vaccine formulations incorporating the post-fusion form of the F protein are highly efficacious in rodent models and a highly efficacious post-fusion F vaccine was also recently reported for RSV, suggesting that the antigenicsite is conserved and of immunologic relevance to the cotton rat model as well. Stable expression of the post-fusion F protein is not trivial and hurdles exist with respect to affordable, consistent production of sufficient quantities of stable, post-fusion F for use in subunit vaccines. However, the availability of the crystal structure and improved strategies to express and stabilize the post-fusion F, significantly enhance the feasibility of a subunit based pre-fusion F vaccine.

In summary, we have shown herein that a single, intranasal administration of the RSV-F nanovaccine elicits mucosal and systemic antiviral immunity, resulting in reduced virus-associated pathology and reduced viral burdens in animals vaccinated at ≤one month of age. The results of our in vivo efficacy studies warrant further evaluation of the RSV-F/G nanovaccine for protection against RSV infection in cotton rat, and have encouraged us to continue to refine the PLGA chemistries and vaccine formulations to optimize their efficacy against RSV infection in neonates.

Claims

A modified RSV F protein, wherein the modified RSV F protein comprises an amino acid sequence having a deletion of 1 to 10 amino acids corresponding to residues 137-146 of SEQ ID NO: 1.
The modified RSV F protein according to claim 1, wherein the modified RSV F protein further comprises an inactivated primary fusion cleavage site.
The modified RSV F protein according to claim 1 or 2, wherein the inactivated primary fusion cleavage site is obtained by mutation of arginine residues at positions 133, 135, and 136 of SEQ ID NO: 1 to glutamine.
The modified RSV F protein according to any one of claims 1-3, wherein the modified RSV F protein comprises or consists of SEQ ID NO: 5.
The modified RSV F protein according to any one of claims 1-4, wherein the modified RSV F protein is a monomeric RSV F protein.
A nucleic sequence encoding the modified RSV F protein according to any one of claims 1-5.
A cell comprising the nucleic sequence according to claim 6.
The cell according to claim 7, wherein the cell is E. coli DH5α cell.
The cell according to claim 8, wherein the E. coli DH5α cell has been deposited with China General Microbiological Culture Collection Center (CGMCC) under the accession number CGMCC No. 25524.
A nanoparticle comprising a viral protein and at least one polymer (s) , wherein the viral protein consists of RSV antigen or antigens, wherein the RSV antigen contains the modified RSV F protein according to any one of claims 1-5.
The nanoparticle according to claim 10, wherein the nanoparticle is immunogenic.
The nanoparticle according to claim 10 or 11, wherein the nanoparticle is a RSV antigen or antigens entrapped within the nanoparticle.
The nanoparticle according to any one of claims 10-12, wherein the polymer is a water soluble, non-adhesive polymer.
The nanoparticle according to any one of claims 10-13, wherein at least one polymer is selected from the group consisting of poly (lactic-co-glycolic acid) , polyethylene glycol, polyethylene oxide, polyalkylene glycol, polyalkylene oxide and polyethylene glycol-poly (lactic-co-glycolic acid) polymer.
The nanoparticle according to any one of claims 10-14, wherein the polymer is poly (lactic-co-glycolic acid) .
The nanoparticle according to any one of claims 10-15, wherein the nanoparticle has an average diameter of about 250 nm to about 600 nm as measured by dynamic light scattering.
A composition comprising: (i) the nanoparticle according to any one of claims 10-16; and (ii) an adjuvant.
The composition according to claim 17, wherein the adjuvant is a Mycobacterium lysate.
The composition according to claim 18, wherein the adjuvant is a Mycobacterium tuberculosis whole cell lysate.
A vaccine comprising the composition according to any one of claims 17-19 and a pharmaceutically acceptable carrier.