Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines.

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Profiling the antibody response of humans protected by immunization with Plasmodium vivax radiation-attenuated sporozoites.

Malaria sterile immunity has been reproducibly induced by immunization with Plasmodium radiation-attenuated sporozoites (RAS). Analyses of sera from RAS-immunized individuals allowed the identification of P. falciparum antigens, such as the circumsporozoite protein (CSP), the basis for the RTS, S and R21Matrix-M vaccines. Similar advances in P. vivax (Pv) vaccination have been elusive. We previously reported 42% (5/12) of sterile protection in malaria-unexposed, Duffy-positive (Fy +) volunteers immunized with PvRAS followed by a controlled human malaria infection (CHMI). Using a custom protein microarray displaying 515 Pv antigens, we found a significantly higher reactivity to PvCSP and one hypothetical protein (PVX_089630) in volunteers protected against P. vivax infection. In mock-vaccinated Fy + volunteers, a strong antibody response to CHMI was also observed. Although the Fy- volunteers immunized with non-irradiated Pv-infected mosquitoes (live sporozoites) did not develop malaria after CHMI, they recognized a high number of antigens, indicating the temporary presence of asexual parasites in peripheral blood. Together, our findings contribute to the understanding of the antibody response to P. vivax infection and allow the identification of novel parasite antigens as vaccine candidates.Trial registration: ClinicalTrials.gov number: NCT01082341.

Impact of sex on humoral immunity with live influenza B virus vaccines in mice.

Influenza B virus (FLUBV) poses a significant infectious threat, with frequent vaccine mismatch limiting its effectiveness. Our previous work investigated the safety and efficacy of modified live attenuated FLUBV vaccines with rearranged genomes (FluB-RAM and FluB-RANS) or a temperature-sensitive PB1 segment with a C-terminal HA tag (FluB-att). In this study, we compared the immune responses of female and male DBA/2J mice vaccinated with these vaccines, including versions containing a chimeric HA segment with an N-terminal IgA-inducing peptide (IGIP). Importantly, both recombinant viruses with and without IGIP remained genetically stable during egg passage. We found that introducing IGIP strengthened vaccine attenuation, particularly for FluB-RAM/IGIP. Prime-boost vaccination completely protected mice against lethal challenge with a homologous FLUBV strain. Notably, recombinant viruses induced robust neutralizing antibody responses (hemagglutination inhibition titers ≥40) alongside antibodies against NA and NP. Interestingly, female mice displayed a consistent trend of enhanced humoral and cross-reactive IgG and IgA responses against HA, NA, and NP compared to male counterparts, regardless of the vaccine used. However, the presence of IGIP generally led to lower anti-HA responses but higher anti-NA and anti-NP responses, particularly of the IgA isotype. These trends were further reflected in mucosal and serological responses two weeks after challenge, with clear distinctions based on sex, vaccine backbone, and IGIP inclusion. These findings hold significant promise for advancing the development of universal influenza vaccines.

Engineering Protein Nanoparticles Functionalized with an Immunodominant Coxiella burnetii Antigen to Generate a Q Fever Vaccine.

Coxiella burnetii is the causative agent of Q fever, for which there is yet to be an FDA-approved vaccine. This bacterial pathogen has both extra- and intracellular stages in its life cycle, and therefore both a cell-mediated (i.e., T lymphocyte) and humoral (i.e., antibody) immune response are necessary for effective eradication of this pathogen. However, most proposed vaccines elicit strong responses to only one mechanism of adaptive immunity, and some can either cause reactogenicity or lack sufficient immunogenicity. In this work, we aim to apply a nanoparticle-based platform toward producing both antibody and T cell immune responses against C. burnetii. We investigated three approaches for conjugation of the immunodominant outer membrane protein antigen (CBU1910) to the E2 nanoparticle to obtain a consistent antigen orientation: direct genetic fusion, high affinity tris-NTA-Ni conjugation to polyhistidine-tagged CBU1910, and the SpyTag/SpyCatcher (ST/SC) system. Overall, we found that the ST/SC approach yielded nanoparticles loaded with the highest number of antigens while maintaining stability, enabling formulations that could simultaneously co-deliver the protein antigen (CBU1910) and adjuvant (CpG1826) on one nanoparticle (CBU1910-CpG-E2). Using protein microarray analyses, we found that after immunization, antigen-bound nanoparticle formulations elicited significantly higher antigen-specific IgG responses than soluble CBU1910 alone and produced more balanced IgG1/IgG2c ratios. Although T cell recall assays from these protein antigen formulations did not show significant increases in antigen-specific IFN-γ production compared to soluble CBU1910 alone, nanoparticles conjugated with a CD4 peptide epitope from CBU1910 generated elevated T cell responses in mice to both the CBU1910 peptide epitope and whole CBU1910 protein. These investigations highlight the feasibility of conjugating antigens to nanoparticles for tuning and improving both humoral- and cell-mediated adaptive immunity against C. burnetii.

Multivalent vaccines demonstrate immunogenicity and protect against Coxiella burnetii aerosol challenge.

Vaccines are among the most cost-effective public health measures for controlling infectious diseases. Coxiella burnetii is the etiological agent of Q fever, a disease with a wide clinical spectrum that ranges from mild symptoms, such as fever and fatigue, to more severe disease, such as pneumonia and endocarditis. The formalin-inactivated whole-cell vaccine Q-VAX® contains hundreds of antigens and confers lifelong protection in humans, but prior sensitization from infection or vaccination can result in deleterious reactogenic responses to vaccination. Consequently, there is great interest in developing non-reactogenic alternatives based on adjuvanted recombinant proteins. In this study, we aimed to develop a multivalent vaccine that conferred protection with reduced reactogenicity. We hypothesized that a multivalent vaccine consisting of multiple antigens would be more immunogenic and protective than a monovalent vaccine owing to the large number of potential protective antigens in the C. burnetii proteome. To address this, we identified immunogenic T and B cell antigens, and selected proteins were purified to evaluate with a combination adjuvant (IVAX-1), with or without C. burnetii lipopolysaccharide (LPS) in immunogenicity studies in vivo in mice and in a Hartley guinea pig intratracheal aerosol challenge model using C. burnetii strain NMI RSA 493. The data showed that multivalent vaccines are more immunogenic than monovalent vaccines and more closely emulate the protection achieved by Q-VAX. Although six antigens were the most immunogenic, we also discovered that multiplexing beyond four antigens introduces detectable reactogenicity, indicating that there is an upper limit to the number of antigens that can be safely included in a multivalent Q-fever vaccine. C. burnetii LPS also demonstrates efficacy as a vaccine antigen in conferring protection in an otherwise monovalent vaccine formulation, suggesting that its addition in multivalent vaccines, as demonstrated by a quadrivalent formulation, would improve protective responses.

Influenza vaccine format mediates distinct cellular and antibody responses in human immune organoids.

Highly effective vaccines elicit specific, robust, and durable adaptive immune responses. To advance informed vaccine design, it is critical that we understand the cellular dynamics underlying responses to different antigen formats. Here, we sought to understand how antigen-specific B and T cells were activated and participated in adaptive immune responses within the mucosal site. Using a human tonsil organoid model, we tracked the differentiation and kinetics of the adaptive immune response to influenza vaccine and virus modalities. Each antigen format elicited distinct B and T cell responses, including differences in their magnitude, diversity, phenotype, function, and breadth. These differences culminated in substantial changes in the corresponding antibody response. A major source of antigen format-related variability was the ability to recruit naive vs. memory B and T cells to the response. These findings have important implications for vaccine design and the generation of protective immune responses in the upper respiratory tract.

Protein Nanoparticle-Mediated Delivery of Recombinant Influenza Hemagglutinin Enhances Immunogenicity and Breadth of the Antibody Response.

The vast majority of seasonal influenza vaccines administered each year are derived from virus propagated in eggs using technology that has changed little since the 1930s. The immunogenicity, durability, and breadth of response would likely benefit from a recombinant nanoparticle-based approach. Although the E2 protein nanoparticle (NP) platform has been previously shown to promote effective cell-mediated responses to peptide epitopes, it has not yet been reported to deliver whole protein antigens. In this study, we synthesized a novel maleimido tris-nitrilotriacetic acid (NTA) linker to couple protein hemagglutinin (HA) from H1N1 influenza virus to the E2 NP, and we evaluated the HA-specific antibody responses using protein microarrays. We found that recombinant H1 protein alone is immunogenic in mice but requires two boosts for IgG to be detected and is strongly IgG1 (Th2) polarized. When conjugated to E2 NPs, IgG2c is produced leading to a more balanced Th1/Th2 response. Inclusion of the Toll-like receptor 4 agonist monophosphoryl lipid A (MPLA) significantly enhances the immunogenicity of H1-E2 NPs while retaining the Th1/Th2 balance. Interestingly, broader homo- and heterosubtypic cross-reactivity is also observed for conjugated H1-E2 with MPLA, compared to unconjugated H1 with or without MPLA. These results highlight the potential of an NP-based delivery of HA for tuning the immunogenicity, breadth, and Th1/Th2 balance generated by recombinant HA-based vaccination. Furthermore, the modularity of this protein-protein conjugation strategy may have utility for future vaccine development against other human pathogens.

Multimericity Amplifies the Synergy of BCR and TLR4 for B Cell Activation and Antibody Class Switching.

Sustained signaling through the B cell antigen receptor (BCR) is thought to occur only when antigen(s) crosslink or disperse multiple BCR units, such as by multimeric antigens found on the surfaces of viruses or bacteria. B cell-intrinsic Toll-like receptor (TLR) signaling synergizes with the BCR to induce and shape antibody production, hallmarked by immunoglobulin (Ig) class switch recombination (CSR) of constant heavy chains from IgM/IgD to IgG, IgA or IgE isotypes, and somatic hypermutation (SHM) of variable heavy and light chains. Full B cell differentiation is essential for protective immunity, where class switched high affinity antibodies neutralize present pathogens, memory B cells are held in reserve for future encounters, and activated B cells also serve as semi-professional APCs for T cells. But the rules that fine-tune B cell differentiation remain partially understood, despite their being essential for naturally acquired immunity and for guiding vaccine development. To address this in part, we have developed a cell culture system using splenic B cells from naive mice stimulated with several biotinylated ligands and antibodies crosslinked by streptavidin reagents. In particular, biotinylated lipopolysaccharide (LPS), a Toll-like receptor 4 (TLR4) agonist, and biotinylated anti-IgM were pre-assembled (multimerized) using streptavidin, or immobilized on nanoparticles coated with streptavidin, and used to active B cells in this precisely controlled, high throughput assay. Using B cell proliferation and Ig class switching as metrics for successful B cell activation, we show that the stimuli are both synergistic and dose-dependent. Crucially, the multimerized immunoconjugates are most active over a narrow concentration range. These data suggest that multimericity is an essential requirement for B cell BCR/TLRs ligands, and clarify basic rules for B cell activation. Such studies highlight the importance in determining the choice of single vs multimeric formats of antigen and PAMP agonists during vaccine design and development.

Magnitude and breadth of antibody cross-reactivity induced by recombinant influenza hemagglutinin trimer vaccine is enhanced by combination adjuvants.

The effects of adjuvants for increasing the immunogenicity of influenza vaccines are well known. However, the effect of adjuvants on increasing the breadth of cross-reactivity is less well understood. In this study we have performed a systematic screen of different toll-like receptor (TLR) agonists, with and without a squalene-in-water emulsion on the immunogenicity of a recombinant trimerized hemagglutinin (HA) vaccine in mice after single-dose administration. Antibody (Ab) cross-reactivity for other variants within and outside the immunizing subtype (homosubtypic and heterosubtypic cross-reactivity, respectively) was assessed using a protein microarray approach. Most adjuvants induced broad IgG profiles, although the response to a combination of CpG, MPLA and AddaVax (termed 'IVAX-1') appeared more quickly and reached a greater magnitude than the other formulations tested. Antigen-specific plasma cell labeling experiments show the components of IVAX-1 are synergistic. This adjuvant preferentially stimulates CD4 T cells to produce Th1>Th2 type (IgG2c>IgG1) antibodies and cytokine responses. Moreover, IVAX-1 induces identical homo- and heterosubtypic IgG and IgA cross-reactivity profiles when administered intranasally. Consistent with these observations, a single-cell transcriptomics analysis demonstrated significant increases in expression of IgG1, IgG2b and IgG2c genes of B cells in H5/IVAX-1 immunized mice relative to naïve mice, as well as significant increases in expression of the IFNγ gene of both CD4 and CD8 T cells. These data support the use of adjuvants for enhancing the breath and durability of antibody responses of influenza virus vaccines.

Distinct SARS-CoV-2 antibody reactivity patterns elicited by natural infection and mRNA vaccination.

We analyzed data from two ongoing COVID-19 longitudinal serological surveys in Orange County, CA., between April 2020 and March 2021. A total of 8476 finger stick blood specimens were collected before and after a vaccination campaign. IgG levels were determined using a multiplex antigen microarray containing antigens from SARS-CoV-2, SARS, MERS, Common CoV, and Influenza. Twenty-six percent of specimens from unvaccinated Orange County residents in December 2020 were SARS-CoV-2 seropositive; out of 852 seropositive individuals 77 had symptoms and 9 sought medical care. The antibody response was predominantly against nucleocapsid (NP), full length, and S2 domain of spike. Anti-receptor binding domain (RBD) reactivity was low and not cross-reactive against SARS S1 or SARS RBD. A vaccination campaign at the University of California Irvine Medical Center (UCIMC) started on December, 2020 and 6724 healthcare workers were vaccinated within 3 weeks. Seroprevalence increased from 13% pre-vaccination to 79% post-vaccination in January, 93% in February, and 99% in March. mRNA vaccination induced higher antibody levels than natural exposure, especially against the RBD domain and cross-reactivity against SARS RBD and S1 was observed. Nucleocapsid protein antibodies can be used to distinguish vaccinees to classify pre-exposure to SARS-CoV-2 Previously infected individuals developed higher antibody titers to the vaccine than non pre-exposed individuals. Hospitalized patients in intensive care with severe disease reach significantly higher antibody levels than mild cases, but lower antibody levels compared to the vaccine. These results indicate that mRNA vaccination rapidly induces a much stronger and broader antibody response than SARS-CoV-2 infection.

Multifunctional Dendronized Polypeptides for Controlled Adjuvanticity.

Vaccination has been playing an important role in treating both infectious and cancerous diseases. Nevertheless, many diseases still lack proper vaccines due to the difficulty to generate sufficient amounts of antigen-specific antibodies or T cells. Adjuvants provide an important route to improve and direct immune responses. However, there are few adjuvants approved clinically and many of them lack the clear structure/adjuvanticity relationship. Here, we synthesized and evaluated a series of dendronized polypeptides (denpols) functionalized with varying tryptophan/histidine (W/H) molar ratios of 0/100, 25/75, 50/50, 75/25, and 100/0 as tunable synthetic adjuvants. The denpols showed structure-dependent inflammasome activation in THP1 monocytic cells and structure-related activation and antigen cross-presentation in vitro in bone marrow-derived dendritic cells. We used the denpols with bacterial pathogen Coxiella burnetii antigens in vivo, which showed both high and tunable adjuvating activities, as demonstrated by the antigen-specific antibody and T cell responses. The denpols are easy to make and scalable, biodegradable, and have highly adjustable chemical structures. Taken together, denpols show great potential as a new and versatile adjuvant platform that allows us to adjust adjuvanticity based on structure-activity correlation with the aim to fine-tune the immune response, thus advancing vaccine development.

Administration of Multivalent Influenza Virus Recombinant Hemagglutinin Vaccine in Combination-Adjuvant Elicits Broad Reactivity Beyond the Vaccine Components.

Combining variant antigens into a multivalent vaccine is a traditional approach used to provide broad coverage against antigenically variable pathogens, such as polio, human papilloma and influenza viruses. However, strategies for increasing the breadth of antibody coverage beyond the vaccine are not well understood, but may provide more anticipatory protection. Influenza virus hemagglutinin (HA) is a prototypic variant antigen. Vaccines that induce HA-specific neutralizing antibodies lose efficacy as amino acid substitutions accumulate in neutralizing epitopes during influenza virus evolution. Here we studied the effect of a potent combination adjuvant (CpG/MPLA/squalene-in-water emulsion) on the breadth and maturation of the antibody response to a representative variant of HA subtypes H1, H5 and H7. Using HA protein microarrays and antigen-specific B cell labelling, we show when administered individually, each HA elicits a cross-reactive antibody profile for multiple variants within the same subtype and other closely-related subtypes (homosubtypic and heterosubtypic cross-reactivity, respectively). Despite a capacity for each subtype to induce heterosubtypic cross-reactivity, broader coverage was elicited by simply combining the subtypes into a multivalent vaccine. Importantly, multiplexing did not compromise antibody avidity or affinity maturation to the individual HA constituents. The use of adjuvants to increase the breadth of antibody coverage beyond the vaccine antigens may help future-proof vaccines against newly-emerging variants.

FluB-RAM and FluB-RANS: Genome Rearrangement as Safe and Efficacious Live Attenuated Influenza B Virus Vaccines.

Influenza B virus (IBV) is considered a major respiratory pathogen responsible for seasonal respiratory disease in humans, particularly severe in children and the elderly. Seasonal influenza vaccination is considered the most efficient strategy to prevent and control IBV infections. Live attenuated influenza virus vaccines (LAIVs) are thought to induce both humoral and cellular immune responses by mimicking a natural infection, but their effectiveness has recently come into question. Thus, the opportunity exists to find alternative approaches to improve overall influenza vaccine effectiveness. Two alternative IBV backbones were developed with rearranged genomes, rearranged M (FluB-RAM) and a rearranged NS (FluB-RANS). Both rearranged viruses showed temperature sensitivity in vitro compared with the WT type B/Bris strain, were genetically stable over multiple passages in embryonated chicken eggs and were attenuated in vivo in mice. In a prime-boost regime in naïve mice, both rearranged viruses induced antibodies against HA with hemagglutination inhibition titers considered of protective value. In addition, antibodies against NA and NP were readily detected with potential protective value. Upon lethal IBV challenge, mice previously vaccinated with either FluB-RAM or FluB-RANS were completely protected against clinical disease and mortality. In conclusion, genome re-arrangement renders efficacious LAIV candidates to protect mice against IBV.

Development of a Novel Live Attenuated Influenza A Virus Vaccine Encoding the IgA-Inducing Protein.

Live attenuated influenza virus (LAIV) vaccines elicit a combination of systemic and mucosal immunity by mimicking a natural infection. To further enhance protective mucosal responses, we incorporated the gene encoding the IgA-inducing protein (IGIP) into the LAIV genomes of the cold-adapted A/Leningrad/134/17/57 (H2N2) strain (caLen) and the experimental attenuated backbone A/turkey/Ohio/313053/04 (H3N2) (OH/04att). Incorporation of IGIP into the caLen background led to a virus that grew poorly in prototypical substrates. In contrast, IGIP in the OH/04att background (IGIP-H1att) virus grew to titers comparable to the isogenic backbone H1att (H1N1) without IGIP. IGIP-H1att- and H1caLen-vaccinated mice were protected against lethal challenge with a homologous virus. The IGIP-H1att vaccine generated robust serum HAI responses in naïve mice against the homologous virus, equal or better than those obtained with the H1caLen vaccine. Analyses of IgG and IgA responses using a protein microarray revealed qualitative differences in humoral and mucosal responses between vaccine groups. Overall, serum and bronchoalveolar lavage samples from the IGIP-H1att group showed trends towards increased stimulation of IgG and IgA responses compared to H1caLen samples. In summary, the introduction of genes encoding immunomodulatory functions into a candidate LAIV can serve as natural adjuvants to improve overall vaccine safety and efficacy.

Subunit Vaccines Using TLR Triagonist Combination Adjuvants Provide Protection Against Coxiella burnetii While Minimizing Reactogenic Responses.

Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii, a designated potential agent of bioterrorism because of its route of transmission, resistance to disinfectants, and low infectious dose. The only vaccine licensed for human use is Q-VAX® (Seqirus, licensed in Australia), a formalin-inactivated whole-cell vaccine, which produces severe local and systemic reactogenic responses in previously sensitized individuals. Accordingly, the U.S. Food and Drug Administration and other regulatory bodies around the world, have been reluctant to approve Q-VAX for widespread use. To obviate these adverse reactions, we prepared recombinant protein subunit vaccine candidates containing purified CBU1910, CBU0307, CBU0545, CBU0612, CBU0891, and CBU1398 proteins and TLR triagonist adjuvants. TLR triagonist adjuvants combine different TLR agonists to enhance immune responses to vaccine antigens. We tested both the protective efficacy and reactogenicity of our vaccine candidates in Hartley guinea pigs using intratracheal infection with live C. burnetii. While all of our candidates showed varying degrees of protection during challenge, local reactogenic responses were significantly reduced for one of our vaccine candidates when compared with a formalin-inactivated whole-cell vaccine. Our findings show that subunit vaccines combined with novel TLR triagonist adjuvants can generate protective immunity to C. burnetii infection while reducing reactogenic responses.

Human Antibody Responses Following Vaccinia Immunization Using Protein Microarrays and Correlation With Cell-Mediated Immunity and Antibody-Dependent Cellular Cytotoxicity Responses.

BACKGROUND: There are limited data regarding immunological correlates of protection for the modified vaccinia Ankara (MVA) smallpox vaccine. METHODS: A total of 523 vaccinia-naive subjects were randomized to receive 2 vaccine doses, as lyophilized MVA given subcutaneously, liquid MVA given subcutaneously (liquid-SC group), or liquid MVA given intradermally (liquid-ID group) 28 days apart. For a subset of subjects, antibody-dependent cellular cytotoxicity (ADCC), interferon-γ release enzyme-linked immunospot (ELISPOT), and protein microarray antibody-binding assays were conducted. Protein microarray responses were assessed for correlations with plaque reduction neutralization titer (PRNT), enzyme-linked immunosorbent assay, ADCC, and ELISPOT results. RESULTS: MVA elicited significant microarray antibody responses to 15 of 224 antigens, mostly virion membrane proteins, at day 28 or 42, particularly WR113/D8L and WR101H3L. In the liquid-SC group, responses to 9 antigens, including WR113/D8L and WR101/H3L, correlated with PRNT results. Three were correlated in the liquid-ID group. No significant correlations were observed with ELISPOT responses. In the liquid-ID group, WR052/F13L, a membrane glycoprotein, correlated with ADCC responses. CONCLUSIONS: MVA elicited antibodies to 15 vaccinia strain antigens representing virion membrane. Antibody responses to 2 proteins strongly increased and significantly correlated with increases in PRNT. Responses to these proteins are potential correlates of protection and may serve as immunogens for future vaccine development. CLINICAL TRIALS REGISTRATION: NCT00914732.

The identification of novel immunogenic antigens as potential Shigella vaccine components.

BACKGROUND: Shigella is a major diarrheal pathogen for which there is presently no vaccine. Whole genome sequencing provides the ability to predict and derive novel antigens for use as vaccines. Here, we aimed to identify novel immunogenic Shigella antigens that could serve as Shigella vaccine candidates, either alone, or when conjugated to Shigella O-antigen. METHODS: Using a reverse vaccinology approach, where genomic analysis informed the Shigella immunome via an antigen microarray, we aimed to identify novel immunogenic Shigella antigens. A core genome analysis of Shigella species, pathogenic and non-pathogenic Escherichia coli, led to the selection of 234 predicted immunogenic Shigella antigens. These antigens were expressed and probed with acute and convalescent serum from microbiologically confirmed Shigella infections. RESULTS: Several Shigella antigens displayed IgG and IgA seroconversion, with no difference in sero-reactivity across by sex or age. IgG sero-reactivity to key Shigella antigens was observed at birth, indicating transplacental antibody transfer. Six antigens (FepA, EmrK, FhuA, MdtA, NlpB, and CjrA) were identified in in vivo testing as capable of producing binding IgG and complement-mediated bactericidal antibody. CONCLUSIONS: These findings provide six novel immunogenic Shigella proteins that could serve as candidate vaccine antigens, species-specific carrier proteins, or targeted adjuvants.

Modeling human adaptive immune responses with tonsil organoids.

Most of what we know about adaptive immunity has come from inbred mouse studies, using methods that are often difficult or impossible to confirm in humans. In addition, vaccine responses in mice are often poorly predictive of responses to those same vaccines in humans. Here we use human tonsils, readily available lymphoid organs, to develop a functional organotypic system that recapitulates key germinal center features in vitro, including the production of antigen-specific antibodies, somatic hypermutation and affinity maturation, plasmablast differentiation and class-switch recombination. We use this system to define the essential cellular components necessary to produce an influenza vaccine response. We also show that it can be used to evaluate humoral immune responses to two priming antigens, rabies vaccine and an adenovirus-based severe acute respiratory syndrome coronavirus 2 vaccine, and to assess the effects of different adjuvants. This system should prove useful for studying critical mechanisms underlying adaptive immunity in much greater depth than previously possible and to rapidly test vaccine candidates and adjuvants in an entirely human system.

Analysis of SARS-CoV-2 antibodies in COVID-19 convalescent blood using a coronavirus antigen microarray.

The current practice for diagnosis of COVID-19, based on SARS-CoV-2 PCR testing of pharyngeal or respiratory specimens in a symptomatic patient at high epidemiologic risk, likely underestimates the true prevalence of infection. Serologic methods can more accurately estimate the disease burden by detecting infections missed by the limited testing performed to date. Here, we describe the validation of a coronavirus antigen microarray containing immunologically significant antigens from SARS-CoV-2, in addition to SARS-CoV, MERS-CoV, common human coronavirus strains, and other common respiratory viruses. A comparison of antibody profiles detected on the array from control sera collected prior to the SARS-CoV-2 pandemic versus convalescent blood specimens from virologically confirmed COVID-19 cases demonstrates near complete discrimination of these two groups, with improved performance from use of antigen combinations that include both spike protein and nucleoprotein. This array can be used as a diagnostic tool, as an epidemiologic tool to more accurately estimate the disease burden of COVID-19, and as a research tool to correlate antibody responses with clinical outcomes.