Induce Neutralizing Antibodies Research Articles

Germline-targeting HIV vaccination induces neutralizing antibodies to the CD4 binding site.

Eliciting potent and broadly neutralizing antibodies (bnAbs) is a major goal in HIV-1 vaccine development. Here, we describe how germline-targeting immunogen BG505 SOSIP germline trimer 1.1 (GT1.1), generated through structure-based design, engages a diverse range of VRC01-class bnAb precursors. A single immunization with GT1.1 expands CD4 binding site (CD4bs)-specific VRC01-class B cells in knock-in mice and drives VRC01-class maturation. In nonhuman primates (NHPs), GT1.1 primes CD4bs-specific neutralizing serum responses. Selected monoclonal antibodies (mAbs) isolated from GT1.1-immunized NHPs neutralize fully glycosylated BG505 virus. Two mAbs, 12C11 and 21N13, neutralize subsets of diverse heterologous neutralization-resistant viruses. High-resolution structures revealed that 21N13 targets the same conserved residues in the CD4bs as VRC01-class and CH235-class bnAbs despite its low sequence similarity (~40%), whereas mAb 12C11 binds predominantly through its heavy chain complementarity-determining region 3. These preclinical data underpin the ongoing evaluation of GT1.1 in a phase 1 clinical trial in healthy volunteers.

Ad26.M.Env ZIKV vaccine protects pregnant rhesus macaques and fetuses against Zika virus infection

At the start of the Zika virus (ZIKV) epidemic in 2015, ZIKV spread across South and Central America, and reached parts of the southern United States placing pregnant women at risk for fetal microcephaly, fetal loss, and other adverse pregnancy outcomes associated with congenital ZIKA syndrome (CZS). For this reason, testing of a safe and efficacious ZIKV vaccine remains a global health priority. Here we report that a single immunization with Ad26.M.Env ZIKV vaccine, when administered prior to conception, fully protects pregnant rhesus macaques from ZIKV viral RNA in blood and tissues with no adverse effects in dams and fetuses. Furthermore, vaccination prevents ZIKV distribution to fetal tissues including the brain. ZIKV associated neuropathology was absent in offspring of Ad26.M.Env vaccinated dams, although pathology was limited in fetuses from non-immunized, challenged dams. Vaccine efficacy is associated with induction of ZIKV neutralizing antibodies in pregnant rhesus macaques. These data suggest the feasibility of vaccine prevention of CZS in humans.

A SARS-CoV-2 peptide antigen purified from bacteria and displayed in a high-density repetitive manner on a virus-like particle could generate anti-SARS-CoV-2 neutralizing antibodies unlike free peptide

SARS-CoV-2 is an enveloped virus. Proteins of the lipid based envelope are not rigidly arranged as compared to non-enveloped viruses lacking lipid based outer layer. Rigidly arranged multivalent display has been reported to be important for potent immune stimulation. We assembled himeric virus-like-particle (VLP) where the core of the particle is composed of the coat protein of Acinetobacter phage. Peptide-based antigen from receptor binding domain (RBD) of SARS-CoV-2 spike protein has been displayed on the coat based VLP matrix using spy-tag/spy-catcher split inteine conjugation system that allows spontaneous, irreversible isopeptide bond formation. Both the matrix as well as peptide have been purified from bacteria which is an easy platform for protein purification. We used the closed state of spike trimer for epitope mapping as this is the conformation of the spike that the immune system visualizes unlike the open state that appears when the virus tries to attach to receptor. Mice based immunization studies were used to test immunogenicity of the antigens. The work demonstrates that peptide-based antigens displayed in high densities can induce neutralizing antibody production unlike free peptide. Careful choice of peptides can deliver better candidates. Also, small size allows easy improvisation. Production in bacteria offers cheaper and robust purification option.

Comparative immunogenicity of monovalent and bivalent adenovirus vaccines carrying spikes of early and late SARS-CoV-2 variants

ABSTRACT The continuous emergence of highly immune-evasive SARS-CoV-2 variants has challenged vaccine efficacy. A vaccine that can provide broad protection is desirable. We evaluated the immunogenicity of a series of monovalent and bivalent adenovirus-vectored vaccines containing the spikes of Wildtype (WT), Beta, Delta, Omicron subvariants BA.1, BA.2, BA.2.12.1, BA.2.13, BA.3, BA.5, BQ.1.1, and XBB. Vaccination in mice using monovalent vaccines elicited the highest neutralizing titers against each self-matched strain, but against other variants were reduced 2− to 73-fold. A bivalent vaccine consisting of WT and BA.5 broadened the neutralizing breadth against pre-Omicron and Omicron subvariants except XBB. Among bivalent vaccines based on the strains before the emergence of XBB, a bivalent vaccine consisting of BA.2 and BA.5 elicited the most potent neutralizing antibodies against Omicron subvariants, including XBB. In mice primed with injected WT vaccine, intranasal booster with a bivalent vaccine containing XBB and BA.5 could elicit broad serum and respiratory mucosal neutralizing antibodies against all late Omicron subvariants, including XBB. In mice that had been sequentially vaccinated with WT and BA.5, intranasal booster with a monovalent XBB vaccine elicited greater serum and mucosal XBB neutralizing antibodies than bivalent vaccines containing XBB. Both monovalent and bivalent XBB vaccines induced neutralizing antibodies against EG.5. Unlike the antibody response, which is highly variant-specific, mice receiving either monovalent or bivalent vaccines elicited comparable T-cell responses against all variants. Furthermore, intranasal but not intramuscular booster induced antigen-specific lung resident T cells. This study provides insights into the design of the COVID-19 vaccine and vaccination strategies.

Safety and antibody responses of Omicron BA.4/5 bivalent booster vaccine among hybrid immunity with diverse vaccination histories: A cohort study

This cohort study, conducted between July and August 2023, evaluated the adverse events (AEs) and immune response to a bivalent mRNA-1273.222 (containing sequences of the original Wuhan-H1 strain and the Omicron BA.4/5 variant) booster vaccine in 122 participants. The study included individuals with diverse vaccination histories, and their responses were assessed based on anti-receptor binding domain (RBD) IgG levels and neutralizing antibodies against the wild-type, Omicron BA.5, and XBB.1.16 variants. Following administration of the BA.4/5 bivalent vaccine, AEs were generally mild to moderate and well-tolerated within a few days. There were no reports of vomiting and no serious AEs or death. The findings demonstrated robust immune responses, with significant increases in anti-RBD IgG levels, particularly in groups that had received 3 –6 doses before the booster dose. The BA.4/5 bivalent booster effectively induced neutralizing antibodies against the vaccine strains, providing robust neutralization, including the XBB.1.16 strain. The study also highlighted that individuals with hybrid immunity, especially those assumed infected with the BA.5 strain or who had been infected twice, showed higher levels of robust neutralizing activity against Omicron XBB.1.16. Overall, these results indicate that the BA.4/5 bivalent booster vaccines can induce potent and good antibody responses in emerging Omicron subvariants, supporting its efficacy as a booster in individuals with diverse vaccination histories.

Why is pet (cat/dog) allergen immunotherapy (AIT) such a controversial topic? Current perspectives and future directions.

Dogs and cats are the most common pets worldwide. In Italy, the prevalence of allergic sensitization to cats and dogs is 16% and 9% respectively. The limited standardization of allergenic extracts, especially for dogs, emphasizes the importance of Component Resolved Diagnosis (CRD) for accurate diagnosis and subsequent prescription of allergen immunotherapy (AIT). However, this low standardization is the main factor contributing to the unsatisfactory clinical efficacy of traditional AIT, AIT with modified allergens, and intralymphatic allergen-specific immunotherapy (ILAIT). Emerging immunological approaches, particularly for controlling the primary cat allergen, show promise but are hindered by high costs (e.g., use of anti-Fel d 1 monoclonal antibodies in humans) or by exclusively targeting Fel d 1 produced by one's own animal (e.g., immunizing cats to induce neutralizing antibodies against Fel d 1 or including an egg product with anti Fel d 1 IgY antibodies in feline diet). Further studies are imperative for standardizing pet allergens, enhancing the efficacy of various AIT modalities, and exploring other immunological approaches, to optimize the relationship between pets and their owners and prevent distressing "forced removals".

Construction and immunogenicity of SARS-CoV-2 virus-like particle expressed by recombinant baculovirus BacMam.

Although existing vaccines have significantly mitigated the impact of the COVID-19 pandemic, none of the vaccines can induce sterilizing immunity. The spike protein is the main component of all approved vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) due primarily to its ability to induce neutralizing antibodies. The conformation of the spike protein in the vaccine formulation should be critical for the efficacy of a vaccine. By way of closely resembling the authentic virions, virus-like particles (VLPs) should render the spike protein in its natural conformation. To this end, we utilized the baculovirus vector, BacMam, to express virus-like particles consisting of the spike, membrane, and envelope proteins of SARS-CoV-2. We demonstrated the immunogenicity of our VLP vaccine with neutralizing activity. Our data warrant further evaluation of the virus-like particles as a vaccine candidate in protecting against virus challenges.

The impact of N-glycans on the immune response of plant-produced SARS-CoV-2 RBD-Fc proteins

Plant-based manufacturing has the advantage of post-translational modifications. While plant-specific N-glycans have been associated with allergic reactions, their effect on the specific immune response upon vaccination is not yet understood. In this study, we produced an RBD-Fc subunit vaccine in both wildtype (WT) and glycoengineered (∆XF) Nicotiana benthamiana plants. The N-glycan analysis: RBD-Fc carrying the ER retention peptide mainly displayed high mannose. When produced in WT RBD-Fc displayed complex-type (GnGnXF) N-glycans. In contrast, ∆XF plants produced RBD-Fc with humanized complex N-glycans that lack potentially immunogenic xylose and core fucose residues (GnGn). The three recombinant RBD-Fc glycovariants were tested. Immunization with any of the RBD-Fc proteins resulted in a similar titer of anti-RBD antibodies in mice. Likewise, antisera from subunit RBD-Fc vaccines also demonstrated comparable neutralization against SARS-CoV-2. Thus, we conclude that N-glycan modifications of the RBD-Fc protein have no impact on their capacity to activate immune responses and induce neutralizing antibody production.

S2 Peptide-Conjugated SARS-CoV-2 Virus-like Particles Provide Broad Protection against SARS-CoV-2 Variants of Concern.

Approved COVID-19 vaccines primarily induce neutralizing antibodies targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike (S) protein. However, the emergence of variants of concern with RBD mutations poses challenges to vaccine efficacy. This study aimed to design a next-generation vaccine that provides broader protection against diverse coronaviruses, focusing on glycan-free S2 peptides as vaccine candidates to overcome the low immunogenicity of the S2 domain due to the N-linked glycans on the S antigen stalk, which can mask S2 antibody responses. Glycan-free S2 peptides were synthesized and attached to SARS-CoV-2 virus-like particles (VLPs) lacking the S antigen. Humoral and cellular immune responses were analyzed after the second booster immunization in BALB/c mice. Enzyme-linked immunosorbent assay revealed the reactivity of sera against SARS-CoV-2 variants, and pseudovirus neutralization assay confirmed neutralizing activities. Among the S2 peptide-conjugated VLPs, the S2.3 (N1135-K1157) and S2.5 (A1174-L1193) peptide-VLP conjugates effectively induced S2-specific serum immunoglobulins. These antisera showed high reactivity against SARS-CoV-2 variant S proteins and effectively inhibited pseudoviral infections. S2 peptide-conjugated VLPs activated SARS-CoV-2 VLP-specific T-cells. The SARS-CoV-2 vaccine incorporating conserved S2 peptides and CoV-2 VLPs shows promise as a universal vaccine capable of generating neutralizing antibodies and T-cell responses against SARS-CoV-2 variants.

Generation of single-round infectious rotavirus with a mutation in the intermediate capsid protein VP6.

Rotavirus causes severe diarrhea in infants. Although live attenuated rotavirus vaccines are available, vaccine-derived infections have been reported, which warrants development of next-generation rotavirus vaccines. A single-round infectious virus is a promising vaccine platform; however, this platform has not been studied extensively in the context of rotavirus. Here, we aimed to develop a single-round infectious rotavirus by impairing the function of the viral intermediate capsid protein VP6. Recombinant rotaviruses harboring mutations in VP6 were rescued using a reverse genetics system. Mutations were targeted at VP6 residues involved in virion assembly. Although the VP6-mutated rotavirus expressed viral proteins, it did not produce progeny virions in wild-type cells; however, the virus did produce progeny virions in VP6-expressing cells. This indicates that the VP6-mutated rotavirus is a single-round infectious rotavirus. Insertion of a foreign gene, and replacement of the VP7 gene segment with that of human rotavirus clinical isolates, was successful. No infectious virions were detected in mice infected with the single-round infectious rotavirus. Immunizing mice with the single-round infectious rotavirus induced neutralizing antibody titers as high as those induced by wild-type rotavirus. Taken together, the data suggest that this single-round infectious rotavirus has potential as a safe and effective rotavirus vaccine. This system is also applicable for generation of safe and orally administrable viral vectors.IMPORTANCERotavirus, a leading cause of acute gastroenteritis in infants, causes an annual estimated 128,500 infant deaths worldwide. Although live attenuated rotavirus vaccines are available, they are replicable and may cause vaccine-derived infections. Thus, development of safe and effective rotavirus vaccine is important. In this study, we report the development of a single-round infectious rotavirus that can replicate only in cells expressing viral VP6 protein. We demonstrated that (1) the single-round infectious rotavirus did not replicate in wild-type cells or in mice; (2) insertion of foreign genes and replacement of the outer capsid gene were possible; and (3) it was as immunogenic as the wild-type virus. Thus, the mutated virus shows promise as a next-generation rotavirus vaccine. The system is also applicable to orally administrable viral vectors, facilitating development of vaccines against other enteric pathogens.

Analysis of binding and authentic virus-neutralizing activities of immune sera induced by various monkeypox virus antigens.

Monkeypox cases continue to increase globally, and there is an urgent need to develop a highly effective vaccine against monkeypox. This study investigated the binding and authentic-virus neutralizing activities of sera from mice immunized with EEV (extracellularly enveloped viruses) antigens B6R and A35R, and IMV (intrinsic material viruses) antigens M1R, A29L, E8L, and H3L against monkeypox virus. The results showed that immunizations of A35R and E8L could only induce lower titers of binding antibodies, in contrast, immunization of M1R induced the highest titers of binding antibodies, while immunization of B6R, H3L, and A29L induced moderate titers of binding antibodies. For the live monkeypox virus neutralization assay, the results showed that immunization with two doses of EEV antigen B6R did not effectively induce humoral immune responses to neutralize monkeypox live virus, immunization with EEV-A35R only induced weak monkeypox-neutralizing antibodies. In contrast, the immunization of the four types of monkeypox virus IMV antigens can all induce neutralizing antibodies against authentic monkeypox virus, among them, A29L and H3L induced the highest neutralizing antibody titers. The results of this study provide important references for the selection of antigens in the development of the next generation of monkeypox vaccines.

Non-cross-reactive epitopes dominate the humoral immune response to COVID-19 vaccination - kinetics of plasma antibodies, plasmablasts and memory B cells.

COVID-19 vaccines are highly effective in inducing protective immunity. While the serum antibody response to COVID-19 vaccination has been studied in depth, our knowledge of the underlying plasmablast and memory B cell (Bmem) responses is still incomplete. Here, we determined the antibody and B cell response to COVID-19 vaccination in a naïve population and contrasted it with the response to a single influenza vaccination in a primed cohort. In addition, we analyzed the antibody and B cell responses against the four endemic human coronaviruses (HCoVs). Measurement of specific plasma IgG antibodies was combined with functional analyses of antibody-secreting plasmablasts and Bmems. SARS-CoV-2- and HCoV-specific IgG antibodies were quantified with an in-house bead-based multiplexed immunoassay. The antibody and B cell responses to COVID-19 vaccination reflected the kinetics of a prime-boost immunization, characterized by a slow and moderate primary response and a faster and stronger secondary response. In contrast, the influenza vaccinees possessed robust immune memory for the vaccine antigens prior to vaccination, and the recall vaccination moderately boosted antibody production and Bmem responses. Antibody levels and Bmem responses waned several months after the 2nd COVID-19 vaccination, but were restored upon the 3rd vaccination. The COVID-19 vaccine-induced antibodies mainly targeted novel, non-cross-reactive S1 epitopes of the viral spike protein, while cross-reactive S2 epitopes were less immunogenic. Booster vaccination not only strongly enhanced neutralizing antibodies against an original SARS-CoV-2 strain, but also induced neutralizing antibodies against the Omicron BA.2 variant. We observed a 100% plasma antibody prevalence against the S1 subunits of HCoVs, which was not affected by vaccination. Overall, by complementing classical serology with a functional evaluation of plasmablasts and memory B cells we provide new insights into the specificity of COVID-19 vaccine-induced antibody and B cell responses.

A low-endotoxic Salmonella enterica Gallinarum serovar delivers infectious bronchitis virus immunogens via a dual-promoter vector system that drives protective immune responses through MHC class-I and -II activation in chickens

An effective vaccine strategy is indispensable against infectious bronchitis virus (IBV) and fowl typhoid (FT), both of which threaten the poultry industry. This study demonstrates a vector system, pJHL270, designed to express antigens in prokaryotic and eukaryotic cells. The vector system stimulates immune responses via synchronized antigen presentation to MHC class-I and -II molecules to produce balanced Th1/Th2 responses. The vaccine antigens were crafted by selecting the consensus sequence of the N-terminal domain of the spike protein (S1-NTD) and a conserved immunogenic region of the nucleocapsid protein (N321-406 aa) from IBV strains circulating in South Korea. The vaccine antigen was cloned and transformed into a live-attenuated Salmonella Gallinarum (SG) strain, JOL2854 (∆lon, ∆cpxR, ∆rfaL, ∆pagL, ∆asd). Western blot analysis confirmed concurrent antigen expression in Salmonella and eukaryotic cells. Oral immunization with the SG-based IBV vaccine construct JOL2918 induced IBV antigen and Salmonella-specific humoral and cell-mediated immune responses in chickens. PBMCs collected from immunized chickens revealed that MHC class-I and -II expression had increased 3.3-fold and 2.5-fold, respectively, confirming MHC activation via bilateral antigen expression and presentation. Immunization induced neutralizing antibodies (NAbs) and reduced the viral load by 2-fold and 2.5-fold in the trachea and lungs, respectively. The immunized chickens exhibited multifaceted humoral, mucosal, and cell-mediated responses via parallel MHC class-I and -II activation as proof of a balanced Th1/Th2 immune response. The level of NAbs, viral load, and gross and histological analyses provide clear evidence that the construct provides protection against IBV and FT.

Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

A Global Perspective on Oral Vaccination of Wildlife against Rabies.

The long-term mitigation of human-domestic animal-wildlife conflicts is complex and difficult. Over the last 50 yr, the primary biomedical concepts and actualized collaborative global field applications of oral rabies vaccination to wildlife serve as one dramatic example that revolutionized the field of infectious disease management of free-ranging animals. Oral vaccination of wildlife occurred in diverse locales within Africa, Eurasia, the Middle East, and North America. Although rabies is not a candidate for eradication, over a billion doses of vaccine-laden baits distributed strategically by hand, at baiting stations, or via aircraft, resulted in widespread disease prevention, control, or local disease elimination among mesocarnivores. Pure, potent, safe, and efficacious vaccines consisted of either modified-live, highly attenuated, or recombinant viruses contained within attractive, edible baits. Since the late 1970s, major free-ranging target species have included coyotes (Canis latrans), foxes (Urocyon cinereoargenteus; Vulpes vulpes), jackals (Canis aureus; Lupulella mesomelas), raccoons (Procyon lotor), raccoon dogs (Nyctereutes procyonoides), and skunks (Mephitis mephitis). Operational progress has occurred in all but the latter species. Programmatic evaluations of oral rabies vaccination success have included: demonstration of biomarkers incorporated within vaccine-laden baits in target species as representative of bait contact; serological measurement of the induction of specific rabies virus neutralizing antibodies, indicative of an immune response to vaccine; and most importantly, the decreasing detection of rabies virus antigens in the brains of collected animals via enhanced laboratory-based surveillance, as evidence of management impact. Although often conceived mistakenly as a panacea, such cost-effective technology applied to free-ranging wildlife represents a real-world, One Health application benefiting agriculture, conservation biology, and public health. Based upon lessons learned with oral rabies vaccination of mesocarnivores, opportunities for future extension to other taxa and additional diseases will have far-reaching, transdisciplinary benefits.

Neutralizing antibody responses to the Delta variant of SARS-CoV-2 following vaccination with Ad5-nCoV (CanSino) in the Mexican population.

During the COVID-19 pandemic, the Ad5-nCoV vaccine was applied to the Mexican population before the WHO approved it. In a transversal study, we compare the CanSino vaccine efficacy and a natural SARS-CoV-2 infection in eliciting neutralizing antibodies against the SARS-CoV-2 Delta variant in Guadalajara, Mexico. Participants between 30-60 years were included in the study and classified into three groups: 1) Natural immunity (unvaccinated), 2) Vaccine-induced immunity (vaccinated individuals without a COVID-19 history), and 3) Natural immunity + vaccine-induced immunity. These groups were matched by age and gender. We assessed the ability of individuals' serum to neutralize the Delta variant and compared the results of the different groups using a neutralization test followed by plaque-forming units. Results showed that 39% of individuals' serum with a history of COVID-19 (natural immunity, Group 1) could not neutralize the Delta variant, compared to 33% in vaccinated individuals without COVID-19 (vaccine immunity, Group 2). In contrast, only 7% of vaccinated individuals with a history of COVID-19 (natural + vaccine immunities) could not neutralize the Delta variant. We concluded that the effectiveness of the Ad5-nCoV vaccine to induce neutralizing antibodies against the Delta variant is comparable to that of natural infection (61% vs. 67%). However, in individuals with both forms of immunity (Group 3), it increased to 93%. Based on these results, despite the Ad5-nCoV vaccine originally being designed as a single-dose regimen, it could be recommended that even those who have recovered from COVID-19 should consider vaccination to boost their immunity against this variant.

A Cocktail of Lipid Nanoparticle-mRNA Vaccines Broaden Immune Responses against β-Coronaviruses in a Murine Model.

Severe acute respiratory syndrome (SARS)-coronavirus (CoV), Middle Eastern respiratory syndrome (MERS)-CoV, and SARS-CoV-2 have seriously threatened human life in the 21st century. Emerging and re-emerging β-coronaviruses after the coronavirus disease 2019 (COVID-19) epidemic remain possible highly pathogenic agents that can endanger human health. Thus, pan-β-coronavirus vaccine strategies to combat the upcoming dangers are urgently needed. In this study, four LNP-mRNA vaccines, named O, D, S, and M, targeting the spike protein of SARS-CoV-2 Omicron, Delta, SARS-CoV, and MERS-CoV, respectively, were synthesized and characterized for purity and integrity. All four LNP-mRNAs induced effective cellular and humoral immune responses against the corresponding spike protein antigens in mice. Furthermore, LNP-mRNA S and D induced neutralizing antibodies against SARS-CoV and SARS-CoV-2, which failed to cross-react with MERS-CoV. Subsequent evaluation of sequential and cocktail immunizations with LNP-mRNA O, D, S, and M effectively elicited broad immunity against SARS-CoV-2 variants, SARS-CoV, and MERS-CoV. A direct comparison of the sequential with cocktail regimens indicated that the cocktail vaccination strategy induced more potent neutralizing antibodies and T-cell responses against heterotypic viruses as well as broader antibody activity against pan-β-coronaviruses. Overall, these results present a potential pan-β-coronavirus vaccine strategy for improved preparedness prior to future coronavirus threats.

Design of hepadnavirus core protein-based chimeric virus-like particles carrying epitopes from respiratory syncytial virus

Respiratory syncytial virus (RSV) is one of the most important pathogens causing respiratory tract infection in humans, especially in infants and the elderly. The identification and structural resolution of the potent neutralizing epitopes on RSV fusion (F) protein enable an “epitope-focused” vaccine design. However, the display of RSV F epitope II on the surface of the widely-used human hepatitis B virus core antigen (HBcAg) has failed to induce neutralizing antibody response in mice. Here, we used the hepadnavirus core protein (HcAg) from different mammalian hosts as scaffolds to construct chimeric virus-like particles (VLPs) presenting the RSV F epitope II. Mouse immunization showed that different HcAg-based chimeric VLPs elicited significantly different neutralizing antibody responses, among which the HcAg derived from roundleaf bat (RBHcAg) is the most immunogenic. Furthermore, RBHcAg was used as the scaffold platform to present multiple RSV F epitopes, and the immunogenicity was further improved in comparison to that displaying a single epitope II. The designed RBHcAg-based multiple-epitope-presenting VLP formulated with MF59-like adjuvant elicited a potent and balanced Th1/Th2 immune response, and offered substantial protection in mice against the challenge of live RSV A2 virus. The designed chimeric VLPs may serve as the potential starting point for developing epitope-focused vaccines against RSV. Our study also demonstrated that RBHcAg is an effective VLP carrier for presenting foreign epitopes, providing a promising platform for epitope-focused vaccine design.

An inactivated whole-virion vaccine for Enterovirus D68 adjuvanted with CpG ODN or AddaVax elicits potent protective immunity in mice

Enterovirus D68 (EV-D68), a pathogen that causes respiratory symptoms, mainly in children, has been implicated in acute flaccid myelitis, which is a poliomyelitis-like paralysis. Currently, there are no licensed vaccines or treatments for EV-D68 infections. Here, we investigated the optimal viral inactivation reagents, vaccine adjuvants, and route of vaccination in mice to optimize an inactivated whole-virion (WV) vaccine against EV-D68. We used formalin, β-propiolactone (BPL), and hydrogen peroxide as viral inactivation reagents and compared their effects on antibody responses. Use of any of these three viral inactivation reagents effectively induced neutralizing antibodies. Moreover, the antibody response induced by the BPL-inactivated WV vaccine was enhanced when adjuvanted with cytosine phosphoguanine oligodeoxynucleotide (CpG ODN) or AddaVax (MF59-like adjuvant), but not with aluminum hydroxide (alum). Consistent with the antibody response results, the protective effect of the inactivated WV vaccine against the EV-D68 challenge was enhanced when adjuvanted with CpG ODN or AddaVax, but not with alum. Further, while the intranasal inactivated WV vaccine induced EV-D68-specific IgA antibodies in the respiratory tract, it was less protective against EV-D68 challenge than the injectable vaccine. Thus, an injectable inactivated EV-D68 WV vaccine prepared with appropriate viral inactivation reagents and an optimal adjuvant is a promising EV-D68 vaccine.

Impact of SARS-CoV-2 spike stability and RBD exposure on antigenicity and immunogenicity

The spike protein (S) of SARS-CoV-2 induces neutralizing antibodies and is the key component of current COVID-19 vaccines. The most efficacious COVID-19 vaccines are genetically-encoded spikes with a double proline substitution in the hinge region to stabilize S in the prefusion conformation (S-2P). A subunit vaccine can be a valuable addition to mRNA and viral vector-based vaccines but requires high stability of spike. In addition, further stabilization of the prefusion conformation of spike might improve immunogenicity. To test this, five spike proteins were designed and characterized, ranging from low to high stability. The immunogenicity of these proteins was assessed in mice, demonstrating that a spike (S-closed-2) with a high melting temperature, which still allowed ACE2 binding, induced the highest neutralization titers against homologous and heterologous strains (up to 16-fold higher than the least stabilized spike). In contrast, the most stable spike variant (S-locked), in which the receptor binding domains (RBDs) were locked in a closed conformation and thus not able to breathe, induced relatively low neutralizing antibody titers against heterologous strains. These data demonstrate that S protein stabilization with RBDs exposing highly conserved epitopes may be needed to increase the immunogenicity of spike proteins for future COVID-19 vaccines.