Eliciting Neutralizing Antibodies Research Articles

Bunyaviruses: Expression of Recombinant Glycoproteins from Insect Cells.

High-density suspension cultures of insect cells offer a scalable and serum-free system for the expression of recombinant proteins. Rift Valley fever virus (RVFV), an arthropod-borne virus spread by mosquitoes, contains two envelop glycoproteins Gn and Gc. These glycoproteins are crucial for eliciting neutralizing antibodies that can offer protection against RVFV infection. Here, we describe the production of individual RVFV glycoproteins and empty RVFV particles using S2 Drosophila insect cells, followed by purification and analysis of the purified proteins.

Hypothermia Exposes Hidden Epitopes in Influenza A Hemagglutinin and Potentiates the Binding of Neutralizing Antibodies

AbstractInfluenza viruses are an important cause of human morbidity and mortality. Influenza uses hemagglutinin, a trimeric surface protein whose monomers are composed of a head and a stalk domain, for immune evasion and cell entry. As the head domain is under constant antigenic drift, eliciting neutralizing antibodies through vaccination against its evolutionarily more conserved stalk domain is a key goal in the treatment of this infection. It is unknown how temperature affects the formation of hemagglutinin‐antibody complexes, particularly with respect to temperatures relevant for the human respiratory tract. Using molecular dynamics simulations and free energy calculations, we determined that bending of hemagglutinin's head domains, an initial step in the process of cell attachment, is present at hypothermia (36 °C), but not at higher temperatures. These conformational changes lead not only to exposure of new epitopes in its stalk domains, but also to enhanced binding of a neutralizing antibody.

Monkeypox virus A29L protein as the target for specific diagnosis and serological analysis

The unexpected monkeypox (Mpox) outbreak has been reported in many non-endemic countries and regions since May 2022. The mutant strains of Mpox virus (MPXV) were found with higher infectivity and greater capability for sustained human-to-human transmission, posing a significant public health threat. MPXV A29L, a protein homolog of vaccinia virus (VACV) A27L, plays an important role in viral attachment to host cell membranes. Therefore, MPXV A29L is considered the diagnostic target and the potential vaccine candidate for eliciting neutralizing antibodies and protective immune responses. In response to the escalating Mpox outbreak, three monoclonal antibodies (mAbs) (2-9B, 3-8G, and 2-5H) targeting the different domains of MPXV A29L have been developed in the study. Among them, 2-5H is highly specific for MPXV A29L without exhibiting cross-reactivity with VACV A27L. The antibody pairing composed of 2-5H and 3-8G has been developed as the lateral flow immunochromatographic assay for specific detection of MPXV A29L. However, these three mAbs were unable to inhibit A29L binding to heparin column or prevent MPXV infection in the neutralization test assays. The results of the serological assays using the truncated A29L fragments as the antigens showed that the Mpox patient sera contained significantly lower levels of antibodies targeting the N-terminal 1–34 residues of A29L, suggesting that the N-terminal portion of A29L is less immunogenic upon natural infection.Key points• MAbs 2-9B, 3-8G, and 2-5H neither interrupted A29L binding to heparin nor neutralized MPXV.• The LFIA composed of 3-8G and 2-5H can specifically distinguish MPXV A29L from VACV A27L.• Mpox patient sera contained lower levels of antibodies targeting the N-terminal portion of A29L.

Enhancing the science in the Global transmission of Nipah virus

Since the Nipah virus's discovery two decades ago, a great deal of knowledge has been applied to the virus's genome, pathology, and wide selectivity of transmission. The highly lethal zoonotic paramyxovirus known as the Nipah virus was first identified in Malaysia in 1998. It's a pathogen that is intimate enough to cause encephalitis caused by the current respiratory virus. The key to controlling discharge is the proactive diagnosis and use of virus management techniques. Only a small number of the 14 bat species found in Malaysia have been identified as vectors of viral transmission, affecting humans, horses, cats, dogs, and pigs. The review provides an explanation of the recent NIV outbreaks in the Philippines, Malaysia, Singapore, Bangladesh, and India. The method of transmission, the preventative and control measures used, and the available evidence following the outbreaks. The virus can also inadvertently result in large financial losses for farmers by seriously sickening animals like pigs. Additionally, it can spread from person to person. Non-invasive ventilation (NIV) is seen in the bronchiole epithelial cells during the early stages of human collapse. The anti-NIV specific IgM was found using the fastest ELISA assay, while the IgG antibody was found using an incidental IgG ELISA. These findings highlight the role that viral glycoproteins play in eliciting neutralizing antibodies, which act as potential vaccines to protect against the illness. Furthermore, there's a chance that these vaccinations will provide cross-protection against similarly related viruses.

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.

Expression of dengue capsid-like particles in silkworm and display of envelope domain III of dengue virus serotype 2

Dengue virus (DENV) is a considerable public health threat affecting millions of people globally. Vaccines for dengue are an important strategy to reduce the disease burden. We expressed capsid (C2) and envelope domain III of dengue virus serotype 2 (2EDIII) separately in the silkworm expression system. We conjugated them employing the monomeric streptavidin (mSA2) and biotin affinity to display the antigenic 2EDIII on the C2-forming capsid-like particle (CLP). Purified 2EDIII-displaying C2 (CLP/2EDIII) was immunogenic in BALB/c mice, eliciting neutralizing antibodies confirmed by a single-round infectious particle (SRIP) neutralization assay. Th1 cytokine levels were upregulated for the CLP/2EDIII group, and the anti-inflammatory IL-10 and pro-inflammatory IL-6 cytokine levels were also raised compared to the 2EDIII and the control groups. Elevated cytokine levels for CLP/2EDIII indicate the importance of displaying the 2EDIII as CLP/2EDIII rather than as an individual subunit. This study is the first to express the C2 protein as self-assembling CLP in vivo and 2EDIII separately in the silkworm expression system and conjugate them to form a monovalent CLP. Thus, this CLP/2EDIII display method may pave the way for an efficient tetravalent dengue vaccine candidate.

Development and evaluation of vaccination strategies for addressing the continuous evolution SARS-CoV-2 based on recombinant trimeric protein technology: Potential for cross-neutralizing activity and broad coronavirus response

Given the significant decline in vaccine efficacy against Omicron, the development of novel vaccines with specific or broad-spectrum effectiveness is paramount. In this study, we formulated four monovalent vaccines based on recombinant spike trimer proteins, along with three bivalent vaccines, and five monovalent vaccines based on recombinant spike proteins. We evaluated the efficacy of different vaccination regimens in eliciting neutralizing antibodies in mice through pseudovirus neutralization assays. Following two doses of primary immunization with D614G, mice received subsequent immunizations with Omicron (BA.1, BA.2, BA.4/5) boosters individually, which led to the generation of broader and more potent cross-neutralizing activity compared to D614G boosters. Notably, the BA.4/5 booster exhibited superior efficacy. Following two doses of primary immunization with Omicron (BA.1, BA.2, BA.4/5), mice were subsequently immunized with one dose of D614G booster which resulted in broader neutralizing activity compared to one dose of Omicron (BA.1, BA.2, or BA.4/5). In unvaccinated mice, full-course immunization with different bivalent vaccines induced broad neutralizing activity against Omicron and pre-Omicron variants, with D614G&BA.4/5 demonstrating superior efficacy. However, compared to other variants, the neutralizing activity against XBB.1.5/1.9.1 is notably reduced. This observation emphasizes the necessity of timely updates to the vaccine antigen composition. Based on these findings and existing studies, we propose a vaccination strategy aimed at preserving the epitope repertoire to its maximum potential: (1) Individuals previously vaccinated or infected with pre-Omicron variants should inoculate a monovalent vaccine containing Omicron components; (2) Individuals who have only been vaccinated or infected with Omicron should be inoculated a monovalent vaccine containing pre-Omicron variants components; (3) Individuals without SARS-CoV-2 infection and vaccination should inoculate a bivalent vaccine comprising both pre-Omicron and Omicron components for primary immunization. Additionally, through cross-inoculation of SARS-CoV-2 D614G spike trimer protein and SARS-CoV-1 spike protein in mice, we preliminarily demonstrated the possibility of cross-reaction between different coronavirus vaccines to produce resistance to the pan-coronavirus.

Immunogen characterization reveals an intrinsic hindrance in eliciting neutralizing antibodies against JN.1 variant

The immune evasion of emerging SARS-CoV-2 variants significantly undermines current vaccination efforts, calling for an updated vaccine composition. To identify optimal booster candidates against circulating JN.1, a panel of variant spikes were characterized. The omicron spikes exhibited reduced plasma membrane expression, accompanied by lower cell-cell fusion but increased viral entry. Regimens with DNA prime-DNA boost or DNA prime-adenoviral vectored vaccine boost by intramuscular immunization elicited neutralizing antibody (NAbs) and Tcell responses against all variants except BA.2.86 and JN.1. Intranasal immunization induced high IgA and NAb titers in bronchoalveolar lavage against all variants except BA.2.86 and JN.1. Tcell responses were generally comparable for all immunogens tested. JN.1 completely escaped NAbs in one immunized cohort, and breakthrough infections marginally boosted antibody titers. Overall, this study indicates intrinsic difficulty in eliciting NAbs against the JN.1 strain, whereas vaccines based on XBB and EG.5.1 are relatively superior in generating cross-reactive NAbs.

The SARS-CoV-2 Spike Protein Receptor-Binding Domain Expressed in Rice Callus Features a Homogeneous Mix of Complex-Type Glycans.

The spike protein receptor-binding domain (RBD) of SARS-CoV-2 is required for the infection of human cells. It is the main target that elicits neutralizing antibodies and also a major component of diagnostic kits. The large demand for this protein has led to the use of plants as a production platform. However, it is necessary to determine the N-glycan structures of an RBD to investigate its efficacy and functionality as a vaccine candidate or diagnostic reagent. Here, we analyzed the N-glycan profile of the RBD produced in rice callus. Of the two potential N-glycan acceptor sites, we found that one was not utilized and the other contained a mixture of complex-type N-glycans. This differs from the heterogeneous mixture of N-glycans found when an RBD is expressed in other hosts, including Nicotiana benthamiana. By comparing the glycosylation profiles of different hosts, we can select platforms that produce RBDs with the most beneficial N-glycan structures for different applications.

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 measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: candidate selection in a preclinical murine model.

In the early COVID-19 pandemic with urgent need for countermeasures, we aimed at developing a replicating viral vaccine using the highly efficacious measles vaccine as vector, a promising technology with prior clinical proof of concept. Building on our successful pre-clinical development of a measles virus (MV)-based vaccine candidate against the related SARS-CoV, we evaluated several recombinant MV expressing codon-optimized SARS-CoV-2 spike glycoprotein. Candidate V591 expressing a prefusion-stabilized spike through introduction of two proline residues in HR1 hinge loop, together with deleted S1/S2 furin cleavage site and additional inactivation of the endoplasmic reticulum retrieval signal, was the most potent in eliciting neutralizing antibodies in mice. After single immunization, V591 induced similar neutralization titers as observed in sera of convalescent patients. The cellular immune response was confirmed to be Th1 skewed. V591 conferred long-lasting protection against SARS-CoV-2 challenge in a murine model with marked decrease in viral RNA load, absence of detectable infectious virus loads, and reduced lesions in the lungs. V591 was furthermore efficacious in an established non-human primate model of disease (see companion article [S. Nambulli, N. Escriou, L. J. Rennick, M. J. Demers, N. L. Tilston-Lunel et al., J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23]). Thus, V591 was taken forward into phase I/II clinical trials in August 2020. Unexpected low immunogenicity in humans (O. Launay, C. Artaud, M. Lachâtre, M. Ait-Ahmed, J. Klein et al., eBioMedicine 75:103810, 2022, https://doi.org/10.1016/j.ebiom.2021.103810) revealed that the underlying mechanisms for resistance or sensitivity to pre-existing anti-measles immunity are not yet understood. Different hypotheses are discussed here, which will be important to investigate for further development of the measles-vectored vaccine platform.IMPORTANCESARS-CoV-2 emerged at the end of 2019 and rapidly spread worldwide causing the COVID-19 pandemic that urgently called for vaccines. We developed a vaccine candidate using the highly efficacious measles vaccine as vector, a technology which has proved highly promising in clinical trials for other pathogens. We report here and in the companion article by Nambulli et al. (J Virol 98:e01762-23, 2024, https://doi.org/10.1128/jvi.01762-23) the design, selection, and preclinical efficacy of the V591 vaccine candidate that was moved into clinical development in August 2020, 7 months after the identification of SARS-CoV-2 in Wuhan. These unique in-human trials of a measles vector-based COVID-19 vaccine revealed insufficient immunogenicity, which may be the consequence of previous exposure to the pediatric measles vaccine. The three studies together in mice, primates, and humans provide a unique insight into the measles-vectored vaccine platform, raising potential limitations of surrogate preclinical models and calling for further refinement of the platform.

Immunogenicity and protective efficacy of inactivated coxsackievirus B4 viral particles

ABSTRACT Coxsackievirus B4 (CVB4) is associated with a range of acute and chronic diseases such as hand, foot, and mouth disease, myocarditis, meningitis, pancreatitis, and type 1 diabetes, affecting millions of young children annually around the world. However, no vaccine is currently available for preventing CVB4 infection. Here, we report the development of inactivated viral particle vaccines for CVB4. Two types of inactivated CVB4 particles were prepared from CVB4-infected cell cultures as vaccine antigens, including F-particle (also called mature virion) consisting of VP1, VP3, VP2, and VP4 subunit proteins, and E-particle (also called empty capsid) which is made of VP1, VP3, and uncleaved VP0. Both the inactivated CVB4 F-particle and E-particle were able to potently elicit neutralizing antibodies in mice, despite slightly lower neutralizing antibody titres seen with the E-particle vaccine after the third immunization. Importantly, we demonstrated that passive transfer of either anti-F-particle or anti-E-particle sera could completely protect the recipient mice from lethal CVB4 challenge. Our study not only defines the immunogenicity and protective efficacy of inactivated CVB4 F-particle and E-particle but also reveals the central role of neutralizing antibodies in anti-CVB4 protective immunity, thus providing important information that may accelerate the development of inactivated CVB4 vaccines.

Approaching the complexity of Crimean-Congo hemorrhagic fever virus serology: A study in swine

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne zoonotic orthonairovirus of public health concern and widespread geographic distribution. Several animal species are known to seroconvert after infection with CCHFV without showing clinical symptoms. The commercial availability of a multi-species ELISA has led to an increase in recent serosurveillance studies as well as in the range of species reported to be exposed to CCHFV in the field, including wild boar (Sus scrofa). However, development and validation of confirmatory serological tests for swine based on different CCHFV antigens or test principles are hampered by the lack of defined control sera from infected and non-infected animals. For the detection of anti-CCHFV antibodies in swine, we established a swine-specific in-house ELISA using a panel of swine sera from CCHFV-free regions and regions with reported CCHFV circulation. We initially screened more than 700 serum samples from wild boar and domestic pigs and observed a correlation of ≃67% between the commercial and the in-house test. From these sera, we selected a panel of 60 samples that were further analyzed in a newly established indirect immunofluorescence assay (iIFA) and virus neutralization test. ELISA-non-reactive samples tested negative. Interestingly, only a subset of samples reactive in both ELISA and iIFA displayed CCHFV-neutralizing antibodies. The observed partial discrepancy between the tests may be explained by different test sensitivities, antibody cross-reactivities or suggests that the immune response to CCHFV in swine is not necessarily associated with eliciting neutralizing antibodies. Overall, this study highlights that meaningful CCHFV serology in swine, and possibly other species, should involve the performance of multiple tests and careful interpretation of the results.

Active immunotherapy for C5a-mediated inflammation using adjuvant-free self-assembled peptide nanofibers

The terminal protein in the complement cascade C5a is a potent inflammatory molecule and chemoattractant that is involved in the pathology of multiple inflammatory diseases including sepsis and arthritis, making it a promising protein to target with immunotherapies. Active immunotherapies, in which patients are immunized against problematic self-molecules and generate therapeutic antibodies as a result, have received increasing interest as an alternative to traditional monoclonal antibody treatments. In previous work, we have designed supramolecular self-assembling peptide nanofibers as active immunotherapies with defined combinations of B- and T-cell epitopes. Herein, the self-assembling peptide Q11 platform was employed to generate a C5a-targeting active immunotherapy. Two of three predicted B-cell epitope peptides from C5a were found to be immunogenic when displayed within Q11 nanofibers, and the nanofibers were capable of reducing C5a serum concentrations following immunization. Contrastingly, C5a's precursor protein C5 maintained its original concentration, promising to minimize side effects heretofore associated with C5-targeted therapies. Immunization protected mice against an LPS-challenge model of sepsis, and it reduced clinical severity in a model of collagen-antibody induced arthritis. Together, this work indicates the potential for targeting terminal complement proteins with active immunotherapies by leveraging the immunogenicity of self-assembled peptide nanomaterials. Statement of significanceChronic inflammatory diseases such as rheumatoid arthritis, psoriasis, and inflammatory bowel disease are currently treated primarily with monoclonal antibodies against key inflammatory mediators. While helpful for many patients, they have high non-response rates, are costly, and commonly fail as anti-drug antibodies are raised by the patient. The approach we describe here explores a fundamentally different treatment paradigm: raising therapeutic antibody responses with an active immunotherapy.We employ innovative supramolecular peptide nanomaterials to elicit neutralizing antibody responses against complement component C5a and demonstrate therapeutic efficacy in preclinical mouse models of sepsis and rheumatoid arthritis. The strategy reported may represent a potential alternative to monoclonal antibody therapies.

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.

Surface-modified measles vaccines encoding oligomeric, prefusion-stabilized SARS-CoV-2 spike glycoproteins boost neutralizing antibody responses to Omicron and historical variants, independent of measles seropositivity.

Serum titers of SARS-CoV-2-neutralizing antibodies (nAbs) correlate well with protection from symptomatic COVID-19 but decay rapidly in the months following vaccination or infection. In contrast, measles-protective nAb titers are lifelong after measles vaccination, possibly due to persistence of the live-attenuated virus in lymphoid tissues. We, therefore, sought to generate a live recombinant measles vaccine capable of driving high SARS-CoV-2 nAb responses. Since previous clinical testing of a live measles vaccine encoding a SARS-CoV-2 spike glycoprotein resulted in suboptimal anti-spike antibody titers, our new vectors were designed to encode prefusion-stabilized SARS-CoV-2 spike glycoproteins, trimerized via an inserted peptide domain, and displayed on a dodecahedral miniferritin scaffold. Additionally, to circumvent the blunting of vaccine efficacy by preformed anti-measles antibodies, we extensively modified the measles surface glycoproteins. Comprehensive in vivo mouse testing demonstrated the potent induction of high titer nAbs in measles-immune mice and confirmed the significant contributions to overall potency afforded by prefusion stabilization, trimerization, and miniferritin display of the SARS-CoV-2 spike glycoprotein. In animals primed and boosted with a measles virus (MeV) vaccine encoding the ancestral SARS-CoV-2 spike, high-titer nAb responses against ancestral virus strains were only weakly cross-reactive with the Omicron variant. However, in primed animals that were boosted with a MeV vaccine encoding the Omicron BA.1 spike, antibody titers to both ancestral and Omicron strains were robustly elevated, and the passive transfer of serum from these animals protected K18-ACE2 mice from infection and morbidity after exposure to BA.1 and WA1/2020 strains. Our results demonstrate that by engineering the antigen, we can develop potent measles-based vaccine candidates against SARS-CoV-2.IMPORTANCEAlthough the live-attenuated measles virus (MeV) is one of the safest and most efficacious human vaccines, a measles-vectored COVID-19 vaccine candidate expressing the SARS-CoV-2 spike failed to elicit neutralizing antibody (nAb) responses in a phase-1 clinical trial, especially in measles-immune individuals. Here, we constructed a comprehensive panel of MeV-based COVID-19 vaccine candidates using a MeV with extensive modifications on the envelope glycoproteins (MeV-MR). We show that artificial trimerization of the spike is critical for the induction of nAbs and that their magnitude can be significantly augmented when the spike protein is synchronously fused to a dodecahedral scaffold. Furthermore, preexisting measles immunity did not abolish heterologous immunity elicited by our vector. Our results highlight the importance of antigen optimization in the development of spike-based COVID-19 vaccines and therapies.

Toxic and antigenic characterization of Peruvian Micrurus surinamensis coral snake venom

Micrurus surinamensis is a semi-aquatic coral snake found in primary forest region and can cause relevant human accidents. In this work we investigated the toxic and antigenic activities of the Peruvian Micrurus surinamensis venom (MsV). We found that MsV show hyaluronidase activity but lack LAAO and PLA2 enzymatic activities. Interestingly, MsV induce edematogenic responses but cannot cause nociceptive effects. Furthermore, MsV can reduce in vitro cell viability in MGSO-3 cell line derived from human breast cancer tissue. To evaluate its antigenic potential, rabbits were immunized with MsV, which proved to be immunogenic. ELISA, immunobloting and in vivo neutralization assays demonstrated that the specific rabbit anti-MsV antivenom is more efficient than the therapeutic Brazilian antivenom in recognizing and neutralizing the lethal activity of MsV. MsV differs in protein profile and biological activities from M. frontalis venom (MfV), used as control, which impairs its recognition and neutralization by Brazilian therapeutic anti-elapidic antivenom. We performed a SPOT immunoassay for the identification of B-cell linear epitopes in the main toxins described for MsV targeted by the elicited neutralizing antibodies previously produced. A membrane containing 15-mer peptides representing the sequences of five 3TFxs and five PLA2s was produced and probed with anti- MsV antibodies. Results revealed important regions in 3FTx toxins for venom neutralization. Identifying the main MsV components and its biological activities can be helpful in guiding the production of antivenoms and in the optimization of treatment for coral snake envenomation in Brazil.

Immunogenicity of novel DNA vaccines encoding receptor-binding domain (RBD) dimer-Fc fusing antigens derived from different SARS-CoV-2 variants of concern.

The continuously emerging of severe acute respiratory syndrome coronavirus-2 variants of concern (VOCs) led to a decline in effectiveness of the first-generation vaccines. Therefore, optimized vaccines and vaccination strategies, which show advantages in protecting against VOCs, are urgently needed. Here we constructed an optimized DNA vaccine plasmid containing built-in CpG adjuvant, and designed vaccine candidates encoding five forms of antigens derived from Wuhan-Hu-1. The results showed that plasmid with receptor binding domain (RBD) dimer-Fc fusing antigen (2RBD-Fc) induced the highest level of RBD-specific IgG and neutralizing antibodies in mice. Then 2dRBD-Fc and 2omRBD-Fc vaccines, respectively derived from delta and omicron VOCs, were constructed. The 2dRBD-Fc induced potent humoral and cellular immune responses, while the immunogenicity of 2omRBD-Fc was low. We also observed that sequential immunization with 2RBD-Fc, 2dRBD-Fc and 2omRBD-Fc effectively elicited neutralizing antibodies against each immunized strain, and RBD-specific T cell responses. To be noted, the Wuhan-Hu-1, delta and omicron neutralizing antibody titers induced by sequential immunization were comparable to that induced by repetitive immunization with 2RBD-Fc, 2dRBD-Fc or 2omRBD-Fc respectively. The results suggest that sequential immunization with DNA vaccines encoding potent antigens derived from different VOCs, may be a promising strategy to elicit immune responses against multiple variants.

A single, improbable B cell receptor mutation confers potent neutralization against cytomegalovirus.

Cytomegalovirus (CMV) is a leading cause of infant hearing loss and neurodevelopmental delay, but there are no clinically licensed vaccines to prevent infection, in part due to challenges eliciting neutralizing antibodies. One of the most well-studied targets for CMV vaccines is the viral fusogen glycoprotein B (gB), which is required for viral entry into host cells. Within gB, antigenic domain 2 site 1 (AD-2S1) is a target of potently neutralizing antibodies, but gB-based candidate vaccines have yet to elicit robust responses against this region. We mapped the genealogy of B cells encoding potently neutralizing anti-gB AD-2S1 antibodies from their inferred unmutated common ancestor (UCA) and characterized the binding and function of early lineage ancestors. Surprisingly, we found that a single amino acid heavy chain mutation A33N, which was an improbable mutation rarely generated by somatic hypermutation machinery, conferred broad CMV neutralization to the non-neutralizing UCA antibody. Structural studies revealed that this mutation mediated key contacts with the gB AD-2S1 epitope. Collectively, these results provide insight into potently neutralizing gB-directed antibody evolution in a single donor and lay a foundation for using this B cell-lineage directed approach for the design of next-generation CMV vaccines.

MRNA vaccines elicit potent neutralization against multiple SARS-CoV-2 Omicron subvariants and other variants of concern.

SARS-CoV-2 variants of concern (VOCs) have shown resistance to vaccines targeting the original virus strain. An mRNA vaccine encoding the spike protein of Omicron BA1 (BA1-S-mRNA) was designed, and its neutralizing activity, with or without the original receptor-binding domain (RBD)-mRNA, was tested against SARS-CoV-2 VOCs. First-dose of BA1-S-mRNA followed by two-boosts of RBD-mRNA elicited potent neutralizing antibodies (nAbs) against pseudotyped and authentic original SARS-CoV-2; pseudotyped Omicron BA1, BA2, BA2.12.1 and BA5 subvariants, and Alpha, Beta, Gamma and Delta VOCs; authentic Omicron BA1 subvariant and Delta VOC. By contrast, other vaccination strategies, including RBD-mRNA first-dose plus BA1-S-mRNA two-boosts, RBD-mRNA or BA1-S-mRNA three-doses, or their combinations, failed to elicit high nAb titers against all of these viruses. Overall, this vaccination strategy was effective for inducing broadly and potent nAbs against multiple SARS-CoV-2 VOCs, particularly Omicron BA5, and may guide the rational design of next-generation mRNA vaccines with greater efficacy against future variants.