Antigen-specific Antibody Responses Research Articles

Gold Nanoparticle Virus-like Particles Presenting SARS-CoV-2 Spike Protein: Synthesis, Biophysical Properties and Immunogenicity in BALB/c Mice

Gold nanoparticles (AuNPs) decorated with antigens have recently emerged as promising tools for vaccine development due to their innate ability to provide stability to antigens and modulate immune responses. In this study, we have engineered deactivated virus-like particles (VLPs) by precisely functionalizing gold cores with coronas comprising the full SARS-CoV-2 spike protein (S). Using BALB/c mice as a model, we investigated the immunogenicity of these S-AuNPs-VLPs. Our results demonstrate that S-AuNPs-VLPs consistently enhanced antigen-specific antibody responses compared to the S protein free in solution. This enhancement included higher binding antibody titers, higher neutralizing capacity of antibodies, and stronger T-cell responses. Compared to the mRNA COVID-19 vaccine, where the S protein is synthesized in situ, S-AuNPs-VLPs induced comparable binding and neutralizing antibody responses, but substantially superior T-cell responses. In conclusion, our study highlights the potential of conjugated AuNPs as an effective antigen-delivery system for protein-based vaccines targeting a broad spectrum of infectious diseases and other emergent viruses.

TRPV1 nociceptors are required to optimize antigen-specific primary antibody responses to novel antigens

BackgroundKey to the advancement of the field of bioelectronic medicine is the identification of novel pathways of neural regulation of immune function. Sensory neurons (termed nociceptors) recognize harmful stimuli and initiate a protective response by eliciting pain and defensive behavior. Nociceptors also interact with immune cells to regulate host defense and inflammatory responses. However, it is still unclear whether nociceptors participate in regulating primary IgG antibody responses to novel antigens.MethodsTo understand the role of transient receptor potential vanilloid 1 (TRPV1)-expressing neurons in IgG responses, we generated TRPV1-Cre/Rosa-ChannelRhodopsin2 mice for precise optogenetic activation of TRPV1 + neurons and TRPV1-Cre/Lox-diphtheria toxin A mice for targeted ablation of TRPV1-expressing neurons. Antigen-specific antibody responses were longitudinally monitored for 28 days.ResultsHere we show that TRPV1 expressing neurons are required to develop an antigen-specific immune response. We demonstrate that selective optogenetic stimulation of TRPV1+ nociceptors during immunization significantly enhances primary IgG antibody responses to novel antigens. Further, mice rendered deficient in TRPV1- expressing nociceptors fail to develop primary IgG antibody responses to keyhole limpet hemocyanin or haptenated antigen.ConclusionThis functional and genetic evidence indicates a critical role for nociceptor TRPV1 in antigen-specific primary antibody responses to novel antigens. These results also support consideration of potential therapeutic manipulation of nociceptor pathways using bioelectronic devices to enhance immune responses to foreign antigens.

Immunogenicity and Protective Capacity of Sugar ABC Transporter Substrate-Binding Protein against Streptococcus suis Serotype 2, 7 and 9 Infection in Mice.

Background:Streptococcus suis (S. suis) is a Gram-positive bacterium that causes substantial disease in pigs. S. suis is also an emerging zoonoses in humans, primarily in Asia, through the consumption of undercooked pork and the handling of infected pig meat as well as carcasses. The complexity of S. suis epidemiology, characterized by the presence of multiple bacterial serotypes and strains with diverse sequence types, identifies a critical need for a universal vaccine with the ability to confer cross-protective immunity. Highly conserved immunogenic proteins are generally considered good candidate antigens for subunit universal vaccines. Methods: In this study, the cross-protection of the sugar ABC transporter substrate-binding protein (S-ABC), a surface-associated immunogenic protein of S. suis, was examined in mice for evaluation as a universal vaccine candidate. Results: S-ABC was shown to be highly conserved, with 97% amino acid sequence identity across 31 S. suis strains deposited in GenBank. Recombinantly expressed S-ABC (rS-ABC) was recognized via rabbit sera specific to S. suis serotype 2. The immunization of mice with rS-ABC induced antigen-specific antibody responses, as well as IFN-γ and IL-4, in multiple organs, including the lungs. rS-ABC immunization conferred high (87.5% and 100%) protection against challenges with S. suis serotypes 2 and 9, demonstrating high cross-protection against these serotypes. Protection, albeit lower (50%), was also observed in mice challenged with S. suis serotype 7. Conclusions: These data identify S-ABC as a promising antigenic target within a universal subunit vaccine against S. suis.

Virus-like particle-based multipathogen vaccine of FMD and SVA elicits balanced and broad protective efficacy in mice and pigs

Inactivated vaccines lack the capability to serologically differentiate between infected and vaccinated animals, thereby impeding the effective eradication of pathogen. Conversely, vaccines based on virus-like particles (VLPs) emulate natural viruses in both size and antigenic structure, presenting a promising alternative to overcome these limitations. As the complexity of swine infectious diseases increases, the increase of vaccine types and doses may intensify the stress response. This exacerbation can lead to diminished productivity, failure of immunization, and elevated costs. Given the critical dynamics of co-infection and the clinically indistinguishable symptoms associated with foot-and-mouth disease virus (FMDV) and senecavirus A (SVA), there is a dire need for an efficacious intervention. To address these challenges, we developed a combined vaccine composed of three distinct VLPs, specifically designed to target SVA and FMDV serotypes O and A. Our research demonstrates that this trivalent VLP vaccine induces antigen-specific and robust serum antibody responses, comparable to those produced by the respective monovalent vaccines. Moreover, the immune sera from the combined VLP vaccine strongly neutralized FMDV type A and O, and SVA, with neutralization titers comparable to those of the individual vaccines, indicating a high level of immunogenic compatibility among the three VLP components. Importantly, the combined VLPs vaccines-immunized sera conferred efficient protection against single or mixed infections with FMDV type A and O, and SVA viruses in pigs. In contrast, individual vaccines could only protect pigs against homologous virus infections and not against heterologous challenges. This study presents a novel combined vaccines candidate against FMD and SVA, and provides new insights for the development of combination vaccines for other viral swine diseases.

The Papain-like Protease Domain of Severe Acute Respiratory Syndrome Coronavirus 2 Conjugated with Human Beta-Defensin 2 and Co1 Induces Mucosal and Systemic Immune Responses against the Virus.

Most of the licensed vaccines against SARS-CoV-2 target spike proteins to induce viral neutralizing antibodies. However, currently prevalent SARS-CoV-2 variants contain many mutations, especially in their spike proteins. The development of vaccine antigens with conserved sequences that cross-react with variants of SARS-CoV-2 is needed to effectively defend against SARS-CoV-2 infection. Given that viral infection is initiated in the respiratory mucosa, strengthening the mucosal immune response would provide effective protection. We constructed a mucosal vaccine antigen using the papain-like protease (PLpro) domain of non-structural protein 3 of SARS-CoV-2. To potentiate the mucosal immune response, PLpro was combined with human beta-defensin 2, an antimicrobial peptide with mucosal immune adjuvant activity, and Co1, an M-cell-targeting ligand. Intranasal administration of the recombinant PLpro antigen conjugate into C57BL/6 and hACE2 knock-in (KI) mice induced antigen-specific T-cell and antibody responses with complement-dependent cytotoxic activity. Viral challenge experiments using the Wuhan and Delta strains of SARS-CoV-2 provided further evidence that immunized hACE2 KI mice were protected against viral challenge infections. Our study shows that PLpro is a useful candidate vaccine antigen against SARS-CoV-2 infection and that the inclusion of human beta-defensin 2 and Co1 in the recombinant construct may enhance the efficacy of the vaccine.

A Quadrivalent mRNA Immunization Elicits Potent Immune Responses against Multiple Orthopoxviral Antigens and Neutralization of Monkeypox Virus in Rodent Models.

The global outbreak of the 2022 monkeypox virus infection of humans and the 2023 documentation of a more virulent monkeypox in the Democratic Republic of the Congo raised public health concerns about the threat of human-to-human transmission of zoonotic diseases. Currently available vaccines may not be sufficient to contain outbreaks of a more transmissible and pathogenic orthopoxvirus. Development of a safe, effective, and scalable vaccine against orthopoxviruses to stockpile for future emergencies is imminent. In this study, we have developed an mRNA vaccine candidate, ALAB-LNP, expressing four vaccinia viral antigens A27, L1, A33, and B5 in tandem in one molecule, and evaluated the vaccine immunogenicity in rodent models. Immunization of animals with the candidate mRNA vaccine induced a potent cellular immune response and long-lasting antigen-specific binding antibody and neutralizing antibody responses against vaccinia virus. Strikingly, the sera from the vaccine-immunized mice cross-reacted with all four homologous antigens of multiple orthopoxviruses and neutralized monkeypox virus in vitro, holding promise for this mRNA vaccine candidate to be used for protection of humans from the infection of monkeypox and other orthopoxvirus.

In situ bio-mineralized Mn nanoadjuvant enhances anti-influenza immunity of recombinant virus-like particle vaccines

Virus like particles (VLPs) have been well recognized as one of the most important vaccine platforms due to their structural similarity to natural viruses to induce effective humoral and cellular immune responses. Nevertheless, lack of viral nucleic acids in VLPs usually leads the vaccine candidates less efficient in provoking innate immune against viral infection. Here, we constructed a biomimetic dual antigen hybrid influenza nanovaccines THM-HA@Mn with robust immunogenicity via in situ synthesizing a stimulator of interferon genes (STING) agonist Mn3O4 inside the cavity of a recombinant Hepatitis B core antigen VLP (HBc VLP) having fused SpyTag and influenza M2e antigen peptides (Tag-HBc-M2e, THM for short), followed by conjugating a recombinant hemagglutinin (rHA) antigen on the surface of the nanoparticles through SpyTag/SpyCatcher ligating. Such inside Mn3O4 immunostimulator-outside rHA antigen design, together with the chimeric M2e antigen on the HBc skeleton, enabled the synthesized hybrid nanovaccines THM-HA@Mn to well imitate the spatial distribution of M2e/HA antigens and immunostimulant in natural influenza virus. In vitro cellular experiments indicated that compared with the THM-HA antigen without Mn3O4 and a mixture vaccine consisting of THM-HA+MnOx, the THM-HA@Mn hybrid nanovaccines showed the highest efficacies in dendritic cells uptake and in promoting BMDC maturation, as well as inducing expression of TNF-α and type I interferon IFN-β. The THM-HA@Mn also displayed the most sustained antigen release at the injection site, the highest efficacies in promoting the DC maturation in lymph nodes and germinal center B cells activation in the spleen of the immunized mice. The co-delivery of immunostimulant and antigens enabled the THM-HA@Mn nanovaccines to induce the highest systemic antigen-specific antibody responses and cellular immunogenicity in mice. Together with the excellent colloid dispersion stability, low cytotoxicity, as well as good biosafety, the synthetic hybrid nanovaccines presented in this study offers a promising strategy to design VLP-based vaccine with robust natural and adaptive immunogenicity against emerging viral pathogens.

Oral administration of DNA alginate nanovaccine induced immune-protection against Helicobacter pylori in Balb/C mice

BackgroundHelicobacter pylori (H. Pylori), is an established causative factor for the development of gastric cancer and the induction of persistent stomach infections that may lead to peptic ulcers. In recent decades, several endeavours have been undertaken to develop a vaccine for H. pylori, although none have advanced to the clinical phase. The development of a successful H. pylori vaccine is hindered by particular challenges, such as the absence of secure mucosal vaccines to enhance local immune responses, the absence of identified antigens that are effective in vaccinations, and the absence of recognized indicators of protection.MethodsThe DNA vaccine was chemically cloned, and the cloning was verified using PCR and restriction enzyme digestion. The efficacy of the vaccination was investigated. The immunogenicity and immune-protective efficacy of the vaccination were assessed in BALB/c mice. This study demonstrated that administering a preventive Alginate/pCI-neo-UreH Nanovaccine directly into the stomach effectively triggered a robust immune response to protect against H. pylori infection in mice.ResultsThe level of immune protection achieved with this nano vaccine was similar to that observed when using the widely accepted formalin-killed H. pylori Hel 305 as a positive control. The Alginate/pCI-neo-UreH Nanovaccine composition elicited significant mucosal and systemic antigen-specific antibody responses and strong intestinal and systemic Th1 responses. Moreover, the activation of IL-17R signaling is necessary for the defensive Th1 immune responses in the intestines triggered by Alginate/pCI-neo-UreH.ConclusionAlginate/pCI-neo-UreH is a potential Nanovaccine for use in an oral vaccine versus H. pylori infection, according to our findings.

Naturally acquired antibodies against Plasmodium vivax pre-erythrocytic stage vaccine antigens inhibit sporozoite invasion of human hepatocytes in vitro

In Plasmodium vivax, the most studied vaccine antigens are aimed at blocking merozoite invasion of erythrocytes and disease development. Very few studies have evaluated pre-erythrocytic (PE) stage antigens. The P. vivax circumsporozoite protein (CSP), is considered the leading PE vaccine candidate, but immunity to CSP is short-lived and variant specific. Thus, there is a need to identify other potential candidates to partner with CSP in a multivalent vaccine to protect against infection and disease. We hypothesize that sporozoite antigens important for host cell infection are considered potential targets. In this study, we evaluated the magnitude and quality of naturally acquired antibody responses to four P. vivax PE antigens: sporozoite surface protein 3 (SSP3), sporozoite protein essential for traversal 1 (SPECT1), cell traversal protein of ookinetes and sporozoites (CelTOS) and CSP in plasma of P. vivax infected patients from Thailand. Naturally acquired antibodies to these antigens were prevalent in the study subjects, but with significant differences in magnitude of IgG antibody responses. About 80% of study participants had antibodies to all four antigens and only 2% did not have antibodies to any of the antigens. Most importantly, these antibodies inhibited sporozoite infection of hepatocytes in vitro. Significant variations in magnitude of antigen-specific inhibitory antibody responses were observed with individual samples. The highest inhibitory responses were observed with anti-CelTOS antibodies, followed by anti-SPECT1, SSP3 and CSP antibodies respectively. These data highlight the vaccine potential of these antigens in protecting against hepatocyte infection and the need for a multi-valent pre-erythrocytic vaccine to prevent liver stage development of P. vivax sporozoites.

N-dihydrogalactochitosan serves as an effective mucosal adjuvant for intranasal vaccine in combination with recombinant viral proteins against respiratory infection

Mucosal vaccinations for respiratory pathogens provide effective protection as they stimulate localized cellular and humoral immunities at the site of infection. Currently, the major limitation of intranasal vaccination is using effective adjuvants capable of withstanding the harsh environment imposed by the mucosa. Herein, we describe the efficacy of using a unique biopolymer, N-dihydrogalactochitosan (GC), as a nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV), an MF-59 equivalent. In contrast to AV, intranasal application of GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. Moreover, GC+S+NC-vaccinated animals were largely resistant to the lethal SARS-CoV-2 challenge and experienced drastically reduced morbidity and mortality, with animal weights and behavior returning to normal 22 days post-infection. In contrast, animals intranasally vaccinated with AV+S+NC experienced severe weight loss, mortality, and respiratory distress, with none surviving beyond 6 days post-infection. Our findings demonstrate that GC can serve as a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses. Statement of significanceWe demonstrated that a unique biopolymer, N-dihydrogalactochitosan (GC), was an effective nasal mucosal vaccine adjuvant against respiratory infections. Specifically, we mixed GC with recombinant SARS-CoV-2 trimeric spike (S) and nucleocapsid (NC) proteins to intranasally vaccinate K18-hACE2 transgenic mice, in comparison with Addavax (AV). In contrast to AV, GC induces a robust, systemic antigen-specific antibody response and increases the number of T cells in the cervical lymph nodes. About 90 % of the GC+S+NC-vaccinated animals survived the lethal SARS-CoV-2 challenge and remained healthy 22 days post-infection, while the AV+S+NC-vaccinated animals experienced severe weight loss and respiratory distress, and all died within 6 days post-infection. Our findings demonstrate that GC is a potent mucosal vaccine adjuvant against SARS-CoV-2 and potentially other respiratory viruses.

A Pvs25 mRNA vaccine induces complete and durable transmission-blocking immunity to Plasmodium vivax

Plasmodium vivax (P. vivax) is the major malaria parasite outside of Africa and no vaccine is available against it. A vaccine that interrupts parasite transmission (transmission-blocking vaccine, TBV) is considered highly desirable to reduce the spread of P. vivax and to accelerate its elimination. However, the development of a TBV against this pathogen has been hampered by the inability to culture the parasite as well as the low immunogenicity of the vaccines developed to date. Pvs25 is the most advanced TBV antigen candidate for P. vivax. However, in previous phase I clinical trials, TBV vaccines based on Pvs25 yielded low antibody responses or had unacceptable safety profiles. As the nucleoside-modified mRNA–lipid nanoparticle (mRNA–LNP) vaccine platform proved to be safe and effective in humans, we generated and tested mRNA–LNP vaccines encoding several versions of Pvs25 in mice. We found that in a prime-boost vaccination schedule, all Pvs25 mRNA–LNP vaccines elicited robust antigen-specific antibody responses. Furthermore, when compared with a Pvs25 recombinant protein vaccine formulated with Montanide ISA-51 adjuvant, the full-length Pvs25 mRNA–LNP vaccine induced a stronger and longer-lasting functional immunity. Seven months after the second vaccination, vaccine-induced antibodies retained the ability to fully block P. vivax transmission in direct membrane feeding assays, whereas the blocking activity induced by the protein/ISA-51 vaccine dropped significantly. Taken together, we report on mRNA vaccines targeting P. vivax and demonstrate that Pvs25 mRNA–LNP outperformed an adjuvanted Pvs25 protein vaccine suggesting that it is a promising candidate for further testing in non-human primates.

Influenza immune imprinting synergizes PEI-HA/CpG nanoparticle vaccine protection against heterosubtypic infection in mice

The first influenza virus infection (imprinting) can lead to long-term immune memory and influence subsequent vaccinations and infections. Previously, we reported a polyethyleneimine (PEI)-Aichi hemagglutinin (HA)/CpG (PHC) nanoparticle with cross-protective potential against homologous and heterologous influenza strains. Here we studied how influenza immune imprinting influences the antibody responses to the PHC vaccination and the protection against heterosubtypic virus challenges. We found that pre-existing virus immunity can maintain or synergize the vaccine-induced antibody titers, depending on the imprinting virus HA phylogenetic group. The HA group 1 virus (PR8, H1N1)-imprinted mice displayed comparable antigen-specific antibody responses to those without imprinting post-PHC vaccination. In contrast, the group 2 virus (Phi, H3N2)-imprinted mice showed significantly more robust and balanced antibodies post-vaccination, conferring complete protection against body weight loss and lung inflammation upon heterosubtypic reassortant A/Shanghai/2/2013 (rSH, H7N9) virus challenge. Our findings suggest that influenza imprinting from the same HA phylogenetic group can synergize subsequent vaccination, conferring heterosubtypic protection.

Mucosal and systemic antigen-specific antibody responses correlate with protection against active tuberculosis in nonhuman primates

Increasing evidence supports that antibodies can protect against active tuberculosis (TB) but knowledge of potentially protective antigens, especially in the airways, is limited. The main objective of this study was to identify antigen-specific airway and systemic immunoglobulin isotype responses associated with the outcome of controlled latent Mycobacterium tuberculosis (Mtb) infection (LTBI) versus uncontrolled infection (TB) in nonhuman primates. In a case-control design, using non-parametric group comparisons with false discovery rate adjustments, we assessed antibodies in 57 cynomolgus macaques which, following low-dose airway Mtb infection, developed either LTBI or TB. We investigated airway and systemic IgG, IgA, and IgM responses in paired bronchoalveolar lavage and plasma samples prior to, two-, and 5-6-months post Mtb infection using an antigen-unbiased approach with Mtb glycan and proteome-wide microarrays. Macaques that developed LTBI (n=36) had significantly increased airway and plasma IgA reactivities to specific arabinomannan (AM) motifs prior to Mtb infection compared to those that developed TB (n=21; p<0.01, q<0.05). Furthermore, LTBI macaques had higher plasma IgG reactivity to protein MTB32A (Rv0125) early post Mtb infection (p<0.05) and increasing airway IgG responses to some proteins over time. Our results support a protective role of pre-existing mucosal (lung) and systemic IgA to specific Mtb glycan motifs, suggesting that prior exposure to nontuberculous mycobacteria could be protective against TB. They further suggest that IgG to Mtb proteins early post infection could provide an additional protective mechanism. These findings could inform TB vaccine development strategies. NIH/NIAID AI117927, AI146329, and AI127173 to JMA.

Production, purification and immunogenicity of Gag virus-like particles carrying SARS-CoV-2 components

The virus-like particle (VLP) platform is a robust inducer of humoral and cellular immune responses; hence, it has been used in vaccine development for several infectious diseases. In the current work, VLPs carrying SARS-CoV-2 Spike (S) protein (Wuhan strain) with an HIV-1 Gag core were produced using suspension HEK 293SF-3F6 cells by transient transfection. The Gag was fused with green fluorescent protein (GFP) for rapid quantification of the VLPs. Five different versions of Gag-Spike VLPs (Gag-S-VLPs) consisting of Gag-S alone or combined with other SARS-CoV-2 components, namely Gag-S-Nucleocapsid (N), Gag-S-Matrix (M), Gag-S-Envelope (E), Gag-S-MEN, along with Gag alone were produced and processed by clarification, nuclease treatment, concentration by tangential flow filtration (TFF) and diafiltration. A pilot mouse study was performed to evaluate the immunogenicity of the Gag-S-VLPs through the measurement of the humoral and/or cellular responses against all the mentioned SARS-CoV-2 components. Antibody response to Spike was observed in all variants. The highest number of Spike-specific IFN-γ + T cells was detected with Gag-S-VLPs. No induction of antigen-specific cellular responses to M, N or E proteins were detected with any of the Gag-S, M, E/or N VLPs tested. Therefore, the Gag-S-VLP, by reason of consistently eliciting strong antigen-specific cellular and antibody responses, was selected for further evaluation. The purification process was improved by replacing the conventional centrifugation by serial microfiltration in the clarification step, followed by Spike-affinity chromatography to get concentrated VLPs with higher purity. Three different doses of Gag-S-VLP in conjunction with two adjuvants (Quil-A or AddaVax) were used to assess the dose-dependent antigen-specific cellular and antibody responses in mice. The Gag-S-VLP adjuvanted with Quil-A resulted in a stronger Spike-specific cellular response compared to that adjuvanted with AddaVax. A strong spike neutralisation activity was observed for all doses, independent of the adjuvant combination.

Dual-Antigen Subunit Vaccine Nanoparticles for Scrub Typhus.

Orientia tsutsugamushi is the causative pathogen of scrub typhus, an acute febrile disease prevalent in the Asia-Pacific region that is spread to people through chigger bites. Despite the emerging threat, there is no currently available vaccine against O. tsutsugamushi. Here, we developed dual-antigen subunit vaccine nanoparticles using recombinant 47 kD and 56 kD proteins, which are immunogenic outer membrane antigens of O. tsutsugamushi. The biocompatible protein vaccine nanoparticles were formed via desolvation of r56 or r47E antigens with acetone, coating with an additional layer of the 56 kD protein, and stabilization with reducible homobifunctional DTSSP and heterobifunctional SDAD crosslinkers. The dual-antigen subunit vaccine nanoparticles significantly improved antigen-specific antibody responses in vaccinated mice. Most importantly, the dual-antigen nanoparticles coated with an additional layer of the 56 kD protein were markedly more immunogenic than soluble antigens or single-antigen nanoparticles in the context of cellular immune responses. Given the significance of cellular immune responses for protection against O. tsutsugamushi, these results demonstrate the potent immunogenicity of dual-layered antigen nanoparticles and their potential as a promising strategy for developing vaccines against scrub typhus.

Capsid-Specific Antibody Responses of Domestic Pigs Immunized with Low-Virulent African Swine Fever Virus.

African swine fever (ASF) is a lethal disease in pigs that has grave socio-economic implications worldwide. For the development of vaccines against the African swine fever virus (ASFV), immunogenic antigens that generate protective immune responses need to be identified. There are over 150 viral proteins-many of which are uncharacterized-and humoral immunity to ASFV has not been closely examined. To profile antigen-specific antibody responses, we developed luciferase-linked antibody capture assays (LACAs) for a panel of ASFV capsid proteins and screened sera from inbred and outbred animals that were previously immunized with low-virulent ASFV before challenge with virulent ASFV. Antibodies to B646L/p72, D117L/p17, M1249L, and E120R/p14.5 were detected in this study; however, we were unable to detect B438L-specific antibodies. Anti-B646L/p72 and B602L antibodies were associated with recovery from disease after challenges with genotype I OUR T88/1 but not genotype II Georgia 2007/1. Antibody responses against M1249L and E120R/p14.5 were observed in animals with reduced clinical signs and viremia. Here, we present LACAs as a tool for the targeted profiling of antigen-specific antibody responses to inform vaccine development.

Antigen-dependent modulation of immune responses to antigen-Fc fusion proteins by Fc-effector functions.

Fc-fusion proteins have been successfully developed for therapeutic purposes, but are also a promising platform for the fast generation and purification of immunogens capable of inducing strong humoral immune responses in preclinical immunization studies. As the Fc-portion of immunoglobulins fused to an antigen confers functional properties of the parental antibody, such as dimerization, binding to Fc-receptors and complement activation, several studies reported that Fc-fusion proteins elicit stronger antigen-specific antibody responses than the unfused antigen. However, dimerization or half-life extension of an antigen have also been described to enhance immunogenicity. To explore the role of Fc-effector functions for the immunogenicity of fusions proteins of viral glycoproteins and Fc fragments, the HIV-1 gp120 and the RBD of SARS-CoV-2 were fused to the wild type muIgG2a Fc fragment or mutants with impaired (LALA-PG) or improved (GASDIE) Fc-effector functions. Immunization of BALB/c mice with DNA vaccines encoding gp120 - Fc LALA-PG induced significantly higher antigen-specific antibody responses than gp120 - Fc WT and GASDIE. In contrast, immunization with DNA vaccines encoding the RBD fused to the same Fc mutants, resulted in comparable anti-RBD antibody levels and similar neutralization activity against several SARS-CoV-2 variants. Depending on the antigen, Fc-effector functions either do not modulate or suppress the immunogenicity of DNA vaccines encoding Fc-antigen fusion proteins.

Reducing cell intrinsic immunity to mRNA vaccine alters adaptive immune responses in mice

The response to mRNA vaccines needs to be sufficient for immune cell activation and recruitment, but moderate enough to ensure efficacious antigen expression. The choice of the cap structure and use of N1-methylpseudouridine (m1Ψ) instead of uridine, which have been shown to reduce RNA sensing by the cellular innate immune system, has led to improved efficacy of mRNA vaccine platforms. Understanding how RNA modifications influence the cell intrinsic immune response may help in the development of more effective mRNA vaccines. In the current study, we compared mRNA vaccines in mice against influenza virus using three different mRNA formats: uridine-containing mRNA (D1-uRNA), m1Ψ-modified mRNA (D1-modRNA), and D1-modRNA with a cap1 structure (cC1-modRNA). D1-uRNA vaccine induced a significantly different gene expression profile to the modified mRNA vaccines, with an up-regulation of Stat1 and RnaseL, and increased systemic inflammation. This result correlated with significantly reduced antigen-specific antibody responses and reduced protection against influenza virus infection compared with D1-modRNA and cC1-modRNA. Incorporation of m1Ψ alone without cap1 improved antibodies, but both modifications were required for the optimum response. Therefore, the incorporation of m1Ψ and cap1 alters protective immunity from mRNA vaccines by altering the innate immune response to the vaccine material.

Design and Purification of Tag/Catcher AP205-Based Capsid Virus-Like Particle Vaccines.

Capsid virus-like particles (cVLPs), assembled from viral coat proteins, are used as therapeutic cargo delivery vehicles as well as molecular scaffolds for display of vaccine antigens. A versatile vaccine platform has been developed based on the Acinetobacter phage AP205 cVLP, which has been shown to significantly improve antigen-specific antibody responses. This modular cVLP platform exploits a split-protein (Tag/Catcher) conjugation system to enable high-density, unidirectional antigen display. Accordingly, protein antigens can be independently expressed and quality-checked prior to conjugation to pre-assembled cVLPs. Here, we describe considerations for the design of vaccine antigens with genetically fused split-protein (Tag or Catcher) binding partners and provide protocols for the expression and purification of corresponding Tag- or Catcher-AP205 cVLPs from E.coli. Finally, we describe a generic protocol for the formulation and quality assessment of experimental/pre-clinical AP205 cVLP-based vaccines.

Self-Assembled Virus-Like Particle Vaccines via Fluorophilic Interactions Enable Infection Mimicry and Immune Protection.

Influenza epidemics persistently threaten global health. Vaccines based on virus-like particles (VLPs), which resemble the native conformation of viruses, have emerged as vaccine candidates. However, the production of VLPs via genetic engineering remains constrained by challenges such as low yields, high costs, and being time consuming. In this study, a novel VLP platform is developed that could mimic infection and confer influenza protection through fluorination-driven self-assembly. The VLPs closely mimick the key steps in viral infection including dendritic cell (DC) attachment and pH-responsive endo-lysosomal escape, which enhances DC maturation and antigen cross-presentation. It is also observed that the VLPs migrate from the injection site to the draining lymph nodes efficiently. Immunization with VLPs triggers both Th1 and Th2 cellular responses, thereby inducing an improved CD8+ T cell response along with strong antigen-specific antibody responses. In several infected mouse models, VLP vaccines ameliorate weight loss, lung virus titers, pulmonary pathologies, and confer full protection against H1N1, H6N2, H9N2, and mixed influenza viruses. Therefore, the results support the potential of VLPs as an effective influenza vaccine with improved immune potency against infection. A methodology to generate VLPs based on fluorophilic interactions, which can be a general approach for development of pathogenic VLPs, is reported.