Elicit Antibody Responses Research Articles

Mosaic and mixed HIV-1 glycoprotein nanoparticles elicit antibody responses to broadly neutralizing epitopes.

An effective human immunodeficiency virus 1 (HIV-1) vaccine will most likely have to elicit broadly neutralizing antibodies (bNAbs) to overcome the sequence diversity of the envelope glycoprotein (Env). So far, stabilized versions of Env, such as SOSIP trimers, have been able to induce neutralizing antibody (NAb) responses, but those responses are mainly strain-specific. Here we attempted to broaden NAb responses by using a multivalent vaccine and applying a number of design improvements. First, we used highly stabilized SOSIP.v9 trimers. Second, we removed any holes in the glycan shields and optimized glycan occupancy to avoid strain-specific glycan hole responses. Third, we selected five sequences from the same clade (B), as we observed previously that combining Env trimers from clade A, B and C did not improve cross-reactive responses, as they might have been too diverse. Fourth, to improve antibody (Ab) responses, the Env trimers were displayed on two-component I53-50 nanoparticles (NPs). Fifth, to favor activation of cross-reactive B cells, the five Env trimers were co-displayed on mosaic NPs. Sixth, we immunized rabbits four times with long intervals between vaccinations. These efforts led to the induction of cross-reactive B cells and cross-reactive binding Ab responses, but we only sporadically detected cross-neutralizing responses. We conclude that stabilized HIV-1 Env trimers that are not modified specifically for priming naive B cells are unable to elicit strong bNAb responses, and infer that sequential immunization regimens, most likely starting with specific germline-targeting immunogens, will be necessary to overcome Env's defenses against the induction of NAbs. The antigens described here could be excellent boosting immunogens in a sequential immunization regimen, as responses to bNAb epitopes were induced.

Abstract A085: Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT

Abstract The goals of this study are to: (a) create a novel soluble bioactive Nectin-4 dimer molecule mimicking its native dimer quaternary structure on the cell surface and (b) evaluate the recombinant Nectin-4 dimer binding potency to its receptor vs the monomer form, so that the dimer can be used as an immunogen and an antigen to increase the potential of cancer therapeutic antibody and vaccine discovery when targeting quaternary structural epitopes. Nectin-4 is a cell adhesion molecule belonging to the nectin family. It's involved in cell-cell junctions and plays a role in cancer progression by influencing cell adhesion, migration, and proliferation. Nectin-4 is overexpressed on various types of tumor cells including breast, lung, ovarian and pancreatic cancers. Nectin-4 has been recently identified as a novel ligand of T cell immunoglobulin and ITIM domain (TIGIT) and interaction between Nectin-4 and TIGIT inhibits NK cell cytotoxicity thus suppressing immune responses, allowing cancer cells to evade immune surveillance. Nectin-4 and Nectin-1 interact in a heterophilic manner, however in the context of cancer, altered expression of Nectin-4 and Nectin-1 can contribute to tumor progression. Nectin-4 is a type 1 single transmembrane protein, and can form homodimers on the cell surface. While therapeutics such as Enfortumab Vedotin, an antibody-drug conjugate, binds Nectin-4 on the surface of cancer cells, mimicking the Nectin-4 dimer quaternary structure can present many more critical opportunities for therapeutic antibody and vaccine discovery. To better mimic the native dimer conformation and quaternary structure, we designed a novel soluble Nectin-4 dimer with 3 extracellular domains fused to a dimer motif at the C-terminus. The Nectin-4 dimer protein was expressed and purified from HEK293 cells and confirmed by SDS-PAGE and Western blot analyses. We characterized the Nectin-4 dimer protein binding potency to TIGIT and Nectin-1 by ELISA. Very interestingly, the results demonstrated that the Nectin-4 dimer binding potency to TIGIT increased dramatically as measured by ELISA, compared to the Nectin-4 monomer protein. Conversely, the Nectin-4 dimer binding to Nectin-1 monomer was significantly decreased, compared to Nectin-4 monomer, as expected. We also studied Nectin-4 dimer immunogenicity using a Nectin-4 dimer DNA immunization to induce potent antibody responses in mice. In summary, these findings imply that the Nectin-4 dimer protein is bioactive and in the desirable conformation, resulting in an increased binding potency to its receptor TIGIT, reduced binding to Nectin-1 (as expected), and eliciting strong antibody responses in mice. This novel Nectin-4 dimer protein can be a very useful molecule for cancer research, therapeutic antibody and vaccine discovery. The Nectin-4 dimer protein can be used to: (i) elicit antibody responses against the dimer quaternary structure, (ii) screen antibodies targeting more conformational epitopes, and (iii) study the Nectin-4 and TIGIT ligand/receptor interactions. Citation Format: Christofer Walsh, Timothy Smith, Mikhail Rashkovskii, Richard Parent, Jean Qiu, Shixia Wang. Engineered novel bioactive Nectin-4 dimer protein significantly enhances the binding of TIGIT [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A085.

Boost and Increased Antibody Breadth Following a Second Dose of PARVAX for SARS-CoV-2 in Mice and Nonhuman Primates.

PARVAX is a genetic vaccine platform based on an adeno-associated vector that has demonstrated to elicit potent, durable, and protective immunity in nonhuman primates (NHPs) after a single dose. Here, we assessed vaccine immunogenicity following a PARVAX prime-boost regimen against SARS-CoV-2. In mice, a low-dose prime followed by a higher-dose boost elicited potent neutralizing antibody responses and distinct cross-reactivity profiles, depending on the antigen used in the booster vaccine. However, the potent neutralizing anti-vector antibody responses developed in mice limited the dose that could be administered as a prime. We further explored the re-administration efficacy in NHPs primed with a SARS-CoV-2 Delta vaccine and boosted with an Omicron BA.1 vaccine at week 15, after the primary response peak antibody levels were reached. The boost elicited an increase in antibodies against several Omicron variants, but no increase was detected in the antibody titers for other variants. The anti-vector responses were low and showed some increased subsequent boosts but generally declined over time. The potent prime vaccination limited the detection of the boosting effect, and therefore, the effect of anti-vector immunity was not fully elucidated. These data show that PARVAX can be effectively re-administered and induce a novel antigenic response.

Immunogenicity of a Rotavirus VP8* Multivalent Subunit Vaccine in Mice.

Rotavirus remains a significant public health threat, especially in low-income countries, where it is the leading cause of severe acute childhood gastroenteritis, contributing to over 128,500 deaths annually. Although the introduction of the Rotarix and RotaTeq vaccines in 2006 marked a milestone in reducing mortality rates, approximately 83,158 preventable deaths persisted, showing ongoing challenges in vaccine accessibility and effectiveness. To address these issues, a novel subcutaneous vaccine formulation targeting multiple rotavirus genotypes has been developed. This vaccine consists of nine VP8* proteins from nine distinct rotavirus genotypes and sub-genotypes (P[4], P[6], P[8]LI, P[8]LIII, P[8]LIV, P[9], P[11], P[14], and P[25]) expressed in E. coli. Two groups of mice were immunized either with a single immunogen, the VP8* from the rotavirus Wa strain (P[8]LI), or with the nonavalent formulation. Preliminary results from mouse immunization studies showed promising outcomes, eliciting antibody responses against six of the nine immunogens. Notably, significantly higher antibody titers against VP8* P[8]LI were observed in the group immunized with the nonavalent vaccine compared to mice specifically immunized against this genotype alone. Overall, the development of parenteral vaccines targeting multiple rotavirus genotypes represents a promising strategy in mitigating the global burden of rotavirus-related morbidity and mortality, offering new avenues for disease prevention and control.

Priming antibody responses to the fusion peptide in rhesus macaques

Immunodominance of antibodies targeting non-neutralizing epitopes and the high level of somatic hypermutation within germinal centers (GCs) required for most HIV broadly neutralizing antibodies (bnAbs) are major impediments to the development of an effective HIV vaccine. Rational protein vaccine design and non-conventional immunization strategies are potential avenues to overcome these hurdles. Here, we report using implantable osmotic pumps to continuously deliver a series of epitope-targeted immunogens to rhesus macaques over the course of six months to prime and elicit antibody responses against the conserved fusion peptide (FP). GC responses and antibody specificities were tracked longitudinally using lymph node fine-needle aspirates and electron microscopy polyclonal epitope mapping (EMPEM), respectively, to show antibody responses to the FP/N611 glycan hole region were primed, although exhibited limited neutralization breadth. Application of cryoEMPEM delineated key residues for on-target and off-target responses that can drive the next round of structure-based vaccine design.

The recombinant vaccine of Lactobacillus plantarum elicits immune protection against H1N1 and H9N2 influenza virus infection

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, resulting in significant economic losses and numerous fatalities. Current vaccines, typically administered through injection, provide limited protection due to the frequent antigenic shift and drift of IAV strains. Therefore, the development of alternative broad-spectrum vaccine strategies is imperative. Lactic acid bacteria (LAB) represent promising candidates for vaccine engineering due to their low cost, high safety profile, and suitability for oral administration. In this study, we identified a strain of Lactobacillus plantarum (Lp) that is resistant to acid and bile salts and capable of colonizing the intestines of mice. Subsequently, we employed the RecE/T gene editing system to integrate headless hemagglutinins (mini-HA) into the genome of Lp, generating Lp-mini-HA-SP. Remarkably, immunization with Lp-mini-HA-SP elicited serum IgG antibody responses and conferred immune protection against H9N2 and H1N1 influenza virus challenges. Collectively, our findings offer a novel approach for the development of orally administered IAV vaccines and hold significant potential for future drug development endeavors.

Field Trials of Live and Inactivated Camelpox Vaccines in Kazakhstan.

An outbreak of camelpox occurred in the Mangistau region of Kazakhstan in 2019. To control the outbreak of camelpox and to prevent its further spread to other regions, camels were vaccinated using live and inactivated camelpox vaccines produced in Kazakhstan. To evaluate the efficacy of these camelpox vaccines in the field, vaccine trials used 172 camels on camel farms in the Beineu district. Of these, 132 camels were vaccinated using a live attenuated camelpox vaccine and 40 camels were vaccinated using an inactivated vaccine to observe immunogenicity and safety. The live vaccine was inoculated into camels by scarification at a dose of 5 × 104 EID50, and the inactivated vaccine was injected intramuscularly at 5 mL twice, with an interval of 35 days. During the safety evaluation, camels administered either vaccine displayed no clinical signs of illness or any adverse effects. Post-vaccination seroconversion demonstrated that the live attenuated vaccine started to elicit antibody responses in some animals as early as day seven, while, by day 28, 99% of vaccinated camels responded. For camels immunized with the inactivated vaccine, seroconversion began on day 21 at low titers ranging from 1:2 to 1:4. Ninety days post vaccination, 77% of the camels demonstrated an immune response that was up to a titer of 1:16. The antibody response waned six months post vaccination in camels vaccinated with two types of vaccine. Nonetheless, both vaccines were 100% effective at preventing clinical disease in vaccinated camels during the camelpox outbreak. All unvaccinated camels became ill, with manifestations of clinical signs characteristic of camelpox. Following these successful field trials in Kazakhstan, a vaccination program for camels, to control camelpox using the domestically produced live attenuated camelpox vaccine, has started.

Epitope-Shared Hapten Enhancing Antibody Polyreactivity for Simultaneous Immunochromatography of Oxyphenisatin Adulterants.

Given the significant threat posed by oxyphenisatin adulterants (OPHs) in weight-loss foods, simultaneous analysis of the OPHs is necessary. Herein, four novel haptens based on the general epitope shared among the OPHs were raised by computer-aided chemical modeling prediction, with the expectation of eliciting antibody responses targeting three of the OPHs. One obtained monoclonal antibody (mAb) showed maximal half-inhibitory concentration (IC50) of 0.40-12.11 ng/mL for OPHs. The key interaction forces responsible for the corecognition of the OPHs were revealed by the intrinsic molecular mechanism. The developed immunochromatography (ICA) indicated a detection capability for screening (CCβ) for OPHs estimated to be 5-600 ng/g in jelly, candy tablets, and oral liquid. Furthermore, the analysis of 15 real samples by our method showed a good correlation with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Our research not only presented a rapid approach for identifying OPHs adulteration but also proposed an effective hapten prediction strategy to enhance antibody polyreactivity.

Heterologous Ad26/Ad5 adenovirus-vectored vaccines elicited SARS-CoV-2-specific antibody responses with potent Fc activities.

Antibodies against the SARS-CoV-2 spike protein are a critical immune determinant for protection against the virus. While virus neutralization is a key function of spike-specific antibodies, antibodies also mediate Fc-dependent activities that can play a role in protection or pathogenesis. This study characterized serum antibody responses elicited after two doses of heterologous adenovirus-vectored (Ad26/ Ad5) vaccines. Vaccine-induced antibody binding titers and Fc-mediated functions decreased over six months, while neutralization titers remained stable. Comparison of antibody isotypes elicited after Ad26/Ad5 vs. LNP-mRNA vaccination and after infection showed that anti-spike IgG1 were dominant and produced to high levels in all groups. The Ad26/Ad5 vaccines also induced IgG4 but not IgG2 and IgG3, whereas the LNP-mRNA vaccines elicited a full Ig spectrum (IgM, IgG1-4, IgA1-2). Convalescent COVID-19 patients had mainly IgM and IgA1 alongside IgG1. Despite these differences, the neutralization potencies against early variants were similar. However, both vaccine groups had antibodies with greater Fc potencies of binding complement and Fcg receptors than the COVID-19 group. The Ad26/Ad5 group also displayed a greater potency of RBD-specific antibody-mediated cellular phagocytosis. Antibodies with distinctive quality were induced by different vaccines and infection. The data imply the utility of different vaccine platforms to elicit antibody responses with fine-tuned Fc activities.

Antibodies against high-risk human papillomavirus proteins as markers for noncervical HPV-related cancers in a Black South African population, according to HIV status.

Human papillomavirus (HPV) proteins may elicit antibody responses in the process toward HPV-related malignancy. However, HPV seroepidemiology in noncervical HPV-related cancers remains poorly understood, particularly in populations with a high prevalence of human immunodeficiency virus (HIV). Using a glutathione S-transferase-based multiplex serology assay, antibodies against E6, E7 and L1 proteins of HPV16 and HPV18 were measured in sera of 535 cases of noncervical HPV-related cancers (anal (n = 104), vulval (n = 211), vaginal (n = 49), penile (n = 37) and oropharyngeal (n = 134)) and 6651 non-infection-related cancer controls, from the Johannesburg Cancer Study that recruited Black South African with newly diagnosed cancer between 1995 and 2016. Logistic and Poisson regression models were used to calculate adjusted odds ratios (aOR) and prevalence ratios (aPR) and 95% confidence intervals (CI) in cases versus controls. HPV16 E6 was more strongly associated with noncervical HPV-related cancers than HPV16 L1 or E7, or HPV18 proteins: anal (females (HPV16 E6 aOR = 11.50;95%CI:6.0-22.2), males (aOR = 10.12;95%CI:4.9-20.8), vulval (aOR = 11.69;95%CI:7.9-17.2), vaginal (aOR = 10.26;95%CI:5.0-21), penile (aOR = 18.95;95%CI:8.9-40), and oropharyngeal (females (aOR = 8.95;95%CI:2.9-27.5), males (aOR = 3.49;95%CI:1.8-7.0)) cancers. HPV16-E6 seropositivity ranged from 24.0% to 35.1% in anal, vulval, vaginal and penile cancer but was significantly lower (11.2%) in oropharyngeal cancer. After adjustment for HIV, prevalence of which increased from 22.2% in 1995-2005 to 54.1% in 2010-2016, HPV16 E6 seropositivity increased by period of diagnosis (aPR for 2010-2016 vs. 1995-2006 = 1.84;95%CI:1.1-3.0). Assuming HPV16 E6 seroprevalence reflects HPV attributable fraction, the proportion of certain noncervical-HPV-related cancers caused by HPV is increasing over time in South Africa. This is expected to be driven by the increasing influence of HIV.

Immunological imprinting shapes the specificity of human antibody responses against SARS-CoV-2 variants

The spike glycoprotein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) continues to accumulate substitutions, leading to breakthrough infections of vaccinated individuals. It remains unclear if exposures to antigenically distant SARS-CoV-2 variants can overcome memory B cell biases established by initial SARS-CoV-2 encounters. We determined the specificity and functionality of antibody and B cell responses following exposure to BA.5 and XBB variants in individuals who received ancestral SARS-CoV-2 mRNA vaccines. BA.5 exposures elicited antibody responses that targeted epitopes conserved between the BA.5 and ancestral spike. XBB exposures also elicited antibody responses that primarily targeted epitopes conserved between the XBB.1.5 and ancestral spike. However, unlike BA.5, a single XBB exposure elicited low frequencies of XBB.1.5-specific antibodies and B cells in some individuals. Pre-existing cross-reactive B cells and antibodies were correlated with stronger overall responses to XBB but weaker XBB-specific responses, suggesting that baseline immunity influences the activation of variant-specific SARS-CoV-2 responses.

ELISA-based detection of immunoglobulins against extracellular vesicles in blood plasma.

Extracellular vesicles (EVs) are intensively investigated for their therapeutic potential and application as drug delivery vehicle. A broad perception of favourable safety profiles and low immunogenicity make EVs an attractive alternative to synthetic nanoparticles. We recently showed that repeated intravenous administration of human cell-derived EVs into pig-tailed macaques unexpectedly elicited antibody responses after three or more injections. This coincided with decreasing EV circulation time, and may thus hamper successful EV-mediated cargo delivery into tissues. Here, we share the custom ELISA protocol that we used to measure such antibody responses. This protocol may help other researchers evaluate immune responses to EV-based therapies in preclinical studies.

Protective efficacy of a Zika purified inactivated virus vaccine candidate during pregnancy in marmosets

Zika virus (ZIKV) infection during pregnancy poses significant threats to maternal and fetal health, leading to intrauterine fetal demise and severe developmental malformations that constitute congenital Zika syndrome (CZS). As such, the development of a safe and effective ZIKV vaccine is a critical public health priority. However, the safety and efficacy of such a vaccine during pregnancy remain uncertain. Historically, the conduct of clinical trials in pregnant women has been challenging. Therefore, clinically relevant animal pregnancy models are in high demand for testing vaccine efficacy. We previously reported that a marmoset pregnancy model of ZIKV infection consistently demonstrated vertical transmission from mother to fetus during pregnancy. Using this marmoset model, we also showed that vertical transmission could be prevented by pre-pregnancy vaccination with Zika purified inactivated virus (ZPIV) vaccine. Here, we further examined the efficacy of ZPIV vaccination during pregnancy. Vaccination during pregnancy elicited virus neutralizing antibody responses that were comparable to those elicited by pre-pregnancy vaccination. Vaccination also reduced placental pathology, viral burden and vertical transmission of ZIKV during pregnancy, without causing adverse effects. These results provide key insights into the safety and efficacy of ZPIV vaccination during pregnancy and demonstrate positive effects of vaccination on the reduction of ZIKV infection, an important advance in preparedness for future ZIKV outbreaks.

Assessment of Immune Responses to a Trivalent Pichinde Virus-Vectored Vaccine Expressing Hemagglutinin Genes from Three Co-Circulating Influenza A Virus Subtypes in Pigs.

Pichinde virus (PICV) can infect several animal species and has been developed as a safe and effective vaccine vector. Our previous study showed that pigs vaccinated with a recombinant PICV-vectored vaccine expressing the hemagglutinin (HA) gene of an H3N2 influenza A virus of swine (IAV-S) developed virus-neutralizing antibodies and were protected against infection by the homologous H3N2 strain. The objective of the current study was to evaluate the immunogenicity and protective efficacy of a trivalent PICV-vectored vaccine expressing HA antigens from the three co-circulating IAV-S subtypes: H1N1, H1N2, and H3N2. Pigs immunized with the trivalent PICV vaccine developed virus-neutralizing (VN) and hemagglutination inhibition (HI) antibodies against all three matching IAV-S. Following challenge infection with the H1N1 strain, five of the six pigs vaccinated with the trivalent vaccine had no evidence of IAV-S RNA genomes in nasal swabs and bronchoalveolar lavage fluid, while all non-vaccinated control pigs showed high number of copies of IAV-S genomic RNA in these two types of samples. Overall, our results demonstrate that the trivalent PICV-vectored vaccine elicits antibody responses against the three targeted IAV-S strains and provides protection against homologous virus challenges in pigs. Therefore, PICV exhibits the potential to be explored as a viral vector for delivering multiple vaccine antigens in swine.

Nanoparticle display of prefusion coronavirus spike elicits S1-focused cross-reactive antibody response against diverse coronavirus subgenera

Multivalent antigen display is a fast-growing area of interest toward broadly protective vaccines. Current nanoparticle-based vaccine candidates demonstrate the ability to confer antibody-mediated immunity against divergent strains of notably mutable viruses. In coronaviruses, this work is predominantly aimed at targeting conserved epitopes of the receptor binding domain. However, targeting conserved non-RBD epitopes could limit the potential for antigenic escape. To explore new potential targets, we engineered protein nanoparticles displaying coronavirus prefusion-stabilized spike (CoV_S-2P) trimers derived from MERS-CoV, SARS-CoV-1, SARS-CoV-2, hCoV-HKU1, and hCoV-OC43 and assessed their immunogenicity in female mice. Monotypic SARS-1 nanoparticles elicit cross-neutralizing antibodies against MERS-CoV and protect against MERS-CoV challenge. MERS and SARS nanoparticles elicit S1-focused antibodies, revealing a conserved site on the S N-terminal domain. Moreover, mosaic nanoparticles co-displaying distinct CoV_S-2P trimers elicit antibody responses to distant cross-group antigens and protect male and female mice against MERS-CoV challenge. Our findings will inform further efforts toward the development of pan-coronavirus vaccines.

A Delta-Omicron Bivalent Subunit Vaccine Elicited Antibody Responses in Mice against Both Ancestral and Variant Strains of SARS-CoV-2.

Continued mutation of the SARS-CoV-2 genome has led to multiple waves of COVID-19 infections, and new variants have continued to emerge and dominate. The emergence of Omicron and its subvariants has substantially increased the infectivity of SARS-CoV-2. RBD genes of the wild-type SARS-CoV-2 strain and the Delta, Omicron BA.1 and Omicron BA.2 variants were used to construct plasmids and express the proteins in glycoengineered Pichia pastoris. A stable 4 L-scale yeast fermentation and purification process was established to obtain high-purity RBD proteins with a complex glycoform N-glycosyl structure that was fucose-free. The RBD glycoproteins were combined with two adjuvants, Al(OH)3 and CpG, which mitigated the typical disadvantage of low immunogenicity associated with recombinant subunit vaccines. To improve the broad-spectrum antiviral activity of the candidate vaccine, Delta RBD proteins were mixed with BA.2 RBD proteins at a ratio of 1:1 and then combined with two adjuvants-Al(OH)3 and CpG-to prepare a bivalent vaccine. The bivalent vaccine effectively induced mice to produce pseudovirus-neutralizing antibodies against SARS-CoV-2 variants, Delta, Beta, and Omicron sublineages BA.1, BA.2, BA.5. The bivalent vaccine could neutralize the authentic wild-type SARS-CoV-2 strain, Delta, BA.1.1, BA.2.2, BA2.3, and BA.2.12.1 viruses, providing a new approach for improving population immunity and delivering broad-spectrum protection under the current epidemic conditions.

A Candidate DNA Vaccine Encoding the Native SARS-CoV-2 Spike Protein Induces Anti-Subdomain 1 Antibodies.

The ideal vaccine against viral infections should elicit antibody responses that protect against divergent strains. Designing broadly protective vaccines against SARS-CoV-2 and other divergent viruses requires insight into the specific targets of cross-protective antibodies on the viral surface protein(s). However, unlike therapeutic monoclonal antibodies, the B-cell epitopes of vaccine-induced polyclonal antibody responses remain poorly defined. Here we show that, through the combination of neutralizing antibody functional responses with B-cell epitope mapping, it is possible to identify unique antibody targets associated with neutralization breadth. The polyclonal antibody profiles of SARS-CoV-2 index-strain-vaccinated rabbits that demonstrated a low, intermediate, or high neutralization efficiency of different SARS-CoV-2 variants of concern (VOCs) were distinctly different. Animals with an intermediate and high cross-neutralization of VOCs targeted fewer antigenic sites on the spike protein and targeted one particular epitope, subdomain 1 (SD1), situated outside the receptor binding domain (RBD). Our results indicate that a targeted functional antibody response and an additional focus on non-RBD epitopes could be effective for broad protection against different SARS-CoV-2 variants. We anticipate that the approach taken in this study can be applied to other viral vaccines for identifying future epitopes that confer cross-neutralizing antibody responses, and that our findings will inform a rational vaccine design for SARS-CoV-2.

Fusion of the molecular adjuvant C3d to cleavage-independent native-like HIV-1 Env trimers improves the elicited antibody response.

An effective HIV vaccine likely requires the elicitation of neutralizing antibodies (NAbs) against multiple HIV-1 clades. The recently developed cleavage-independent native flexibly linked (NFL) envelope (Env) trimers exhibit well-ordered conformation and elicit autologous tier 2 NAbs in multiple animal models. Here, we investigated whether the fusion of molecular adjuvant C3d to the Env trimers can improve B- cell germinal center (GC) formation and antibody responses. To generate Env-C3d trimers, we performed a glycine-serine- based (G4S) flexible peptide linker screening and identified a linker range that allowed native folding. A 30-60- amino- acid- long linker facilitates Env-to-C3d association and achieves the secretion of well-ordered trimers and the structural integrity and functional integrity of Env and C3d. The fusion of C3d did not dramatically affect the antigenicity of the Env trimers and enhanced the ability of the Env trimers to engage and activate B cells in vitro. In mice, the fusion of C3d enhanced germinal center formation, the magnitude of Env-specific binding antibodies, and the avidity of the antibodies in the presence of an adjuvant. The Sigma Adjuvant System (SAS) did not affect the trimer integrity in vitro but contributed to altered immunogenicity in vivo, resulting in increased tier 1 neutralization, likely by increased exposure of variable region 3 (V3). Taken together, the results indicate that the fusion of the molecular adjuvant, C3d, to the Env trimers improves antibody responses and could be useful for Env-based vaccines against HIV.

The Gold Nanoparticles-Functionalized with the Synthetic PADRE^S2P6 Peptide Can Be Useful for SARS-CoV-2 Detection

Conjugation of bioactive peptides to nanomaterials is a promising approach for a variety of biomedical uses. Indeed, we assumed that gold nanoparticles (AuNPs) functionalized with synthetic viral peptides represent a promising strategy to elicit antibody response against zoonotic coronavirus SARS-CoV-2 responsible for pandemic COVID-19 disease. Two specific linear B-cell epitopes namely S1P4 and S2P6 have been recently identified in the SARS-CoV-2 spike protein expressed by the COVID-19 mRNA BNT162 vaccine of Pfizer-BioNTech and marketed under the brand name Comirnaty. The present study aimed at investigating the immunogenic potential of AuNPs functionalized with synthetic PADRE^S1P4 and PADRE^S2P6 peptides in a mouse model. The AuNPs were synthesized using an environmentally friendly process. In both synthetic PADRE^S1P4 and PADRE^S2P6 peptides, the SARS-CoV-2 spike antibody epitope is preceded by a polybasic sequence and the T-helper cell response activator PADRE. A thiol-terminated polyethylene glycol was used to decorate AuNP surface with the synthetic peptides. The AuNPs-peptide conjugates were inoculated without any adjuvant to adult BALB/c mice by intramuscular route in a prime-boost schedule. The AuNPs functionalized with the PADRE^S2P6 peptide but not the PADRE^S1P4 peptide were efficient to elicit antibody production of relevant specificity against the SARS-CoV-2 spike protein. The ability of PADRE^S2P6 peptide-reactive antibodies to recognize SARS-CoV-2 variants opens important perspectives for AuNP-peptide conjugates as potential serological tools to support the surveillance of wildlife-origin coronaviruses.

Amount of antigen, T follicular helper cells and affinity of founder cells shape the diversity of germinal center B cells: A computational study.

A variety of B cell clones seed the germinal centers, where a selection stringency expands the fitter clones to generate higher affinity antibodies. However, recent experiments suggest that germinal centers often retain a diverse set of B cell clones with a range of affinities and concurrently carry out affinity maturation. Amid a tendency to flourish germinal centers with fitter clones, how several B cell clones with differing affinities can be concurrently selected remains poorly understood. Such a permissive selection may allow non-immunodominant clones, which are often rare and of low-affinity, to somatically hypermutate and result in a broad and diverse B cell response. How the constituent elements of germinal centers, their quantity and kinetics may modulate diversity of B cells, has not been addressed well. By implementing a state-of-the-art agent-based model of germinal center, here, we study how these factors impact temporal evolution of B cell clonal diversity and its underlying balance with affinity maturation. While we find that the extent of selection stringency dictates clonal dominance, limited antigen availability on follicular dendritic cells is shown to expedite the loss of diversity of B cells as germinal centers mature. Intriguingly, the emergence of a diverse set of germinal center B cells depends on high affinity founder cells. Our analysis also reveals a substantial number of T follicular helper cells to be essential in balancing affinity maturation with clonal diversity, as a low number of T follicular helper cells impedes affinity maturation and also contracts the scope for a diverse B cell response. Our results have implications for eliciting antibody responses to non-immunodominant specificities of the pathogens by controlling the regulators of the germinal center reaction, thereby pivoting a way for vaccine development to generate broadly protective antibodies.