Waning immunity and IgG4 responses following bivalent mRNA boosting.

Messenger RNA (mRNA) vaccines were highly effective against the ancestral SARS-CoV-2 strain, but the efficacy of bivalent mRNA boosters against XBB variants was substantially lower. Here, we show limited durability of neutralizing antibody (NAb) responses against XBB variants and isotype switching to immunoglobulin G4 (IgG4) responses following bivalent mRNA boosting. Bivalent mRNA boosting elicited modest XBB.1-, XBB.1.5-, and XBB.1.16-specific NAbs that waned rapidly within 3 months. In contrast, bivalent mRNA boosting induced more robust and sustained NAbs against the ancestral WA1/2020 strain, suggesting immune imprinting. Following bivalent mRNA boosting, serum antibody responses were primarily IgG2 and IgG4 responses with poor Fc functional activity. In contrast, a third monovalent mRNA immunization boosted all isotypes including IgG1 and IgG3 with robust Fc functional activity. These data show substantial immune imprinting for the ancestral spike and isotype switching to IgG4 responses following bivalent mRNA boosting, with important implications for future booster designs and boosting strategies.

Waning Immunity Against XBB.1.5 Following Bivalent mRNA Boosters.

The SARS-CoV-2 Omicron variant has continued to evolve. XBB is a recombinant between two BA.2 sublineages, XBB.1 includes the G252V mutation, and XBB.1.5 includes the G252V and F486P mutations. XBB.1.5 has rapidly increased in frequency and has become the dominant virus in New England. The bivalent mRNA vaccine boosters have been shown to increase neutralizing antibody (NAb) titers to multiple variants, but the durability of these responses remains to be determined. We assessed humoral and cellular immune responses in 30 participants who received the bivalent mRNA boosters and performed assays at baseline prior to boosting, at week 3 after boosting, and at month 3 after boosting. Our data demonstrate that XBB.1.5 substantially escapes NAb responses but not T cell responses after bivalent mRNA boosting. NAb titers to XBB.1 and XBB.1.5 were similar, suggesting that the F486P mutation confers greater transmissibility but not increased immune escape. By month 3, NAb titers to XBB.1 and XBB.1.5 declined essentially to baseline levels prior to boosting, while NAb titers to other variants declined less strikingly.

Altered Cytokine Production in Human Intervillous Blood T Cells in Preeclampsia.

Conventional and regulatory T cells (Treg) are dynamic mediators of maternal immune tolerance to the developing feto-placental unit. Functional evaluation of T cells at the maternal-fetal interface is crucial to elucidate the immunologic basis of obstetric complications. Our objective was to define the T cell phenotype and function of uterine intervillous blood (IVB) in pregnancy with and without preeclampsia. We hypothesize that preeclampsia is associated with impaired immune tolerance and a pro-inflammatory uterine T cell microenvironment. In this cross-sectional study, maternal peripheral blood (PB) and uterine IVB (obtained from the surgical sponge used to clean the placental bed during cesarean delivery) were collected from participants with and without preeclampsia. Proportion, activation, and cytokine production of T cell subsets were quantified by flow cytometry. T cell parameters were compared by tissue source and by preeclampsia status. Sixty participants, 26 with preeclampsia, were included. Induced Treg made up a greater proportion of IVB T cells compared to PB and had greater cytokine-producing capacity. Preeclampsia was associated with increased ratio of pro-inflammatory IL-17α to suppressive IL-10 cytokine production by CD4 T cell subsets in IVB, but not in PB. Human uterine IVB is composed of activated, cytokine-producing T cell subsets distinct from maternal PB. Preeclampsia is associated with a pro-inflammatory IVB profile, with increased IL-17α /IL-10 ratio in all CD4 T cell subsets. IVB sampling is a useful tool for investigating human T cell biology at the maternal-fetal interface that may inform immunotherapeutic strategies for preeclampsia.

Immunogenicity of the BA.5 Bivalent mRNA Vaccine Boosters.

Waning immunity following mRNA vaccination and the emergence of SARS-CoV-2 variants has led to reduced mRNA vaccine efficacy against both symptomatic infection and severe disease. Bivalent mRNA boosters expressing the Omicron BA.5 and ancestral WA1/2020 Spike proteins have been developed and approved, because BA.5 is currently the dominant SARS-CoV-2 variant and substantially evades neutralizing antibodies (NAbs). Our data show that BA.5 NAb titers were comparable following monovalent and bivalent mRNA boosters.

Durability of Heterologous and Homologous COVID-19 Vaccine Boosts.

Importance: Antibody responses elicited by current messenger RNA (mRNA) COVID-19 vaccines decline rapidly and require repeated boosting. Objective: To evaluate the immunogenicity and durability of heterologous and homologous prime-boost regimens involving the adenovirus vector vaccine Ad26.COV2.S and the mRNA vaccine BNT162b2. Design, Setting, and Participants: In this cohort study at a single clinical site in Boston, Massachusetts, 68 individuals who were vaccinated at least 6 months previously with 2 immunizations of BNT162b2 were boosted with either Ad26.COV2.S or BNT162b2. Enrollment of participants occurred from August 12, 2021, to October 25, 2021, and this study involved 4 months of follow-up. Data analysis was performed from November 2021 to February 2022. Exposures: Participants who were previously vaccinated with BNT162b2 received a boost with either Ad26.COV2.S or BNT162b2. Main Outcomes and Measures: Humoral immune responses were assessed by neutralizing, binding, and functional antibody responses for 16 weeks following the boost. CD8+ and CD4+ T-cell responses were evaluated by intracellular cytokine staining assays. Results: Among 68 participants who were originally vaccinated with BNT162b2 and boosted with Ad26.COV2.S (41 participants; median [range] age, 36 [23-84] years) or BNT162b2 (27 participants; median [range] age, 35 [23-76] years), 56 participants (82%) were female, 7 (10%) were Asian, 4 (6%) were Black, 4 (6%) were Hispanic or Latino, 3 (4%) were more than 1 race, and 53 (78%) were White. Both vaccines were found to be associated with increased humoral and cellular immune responses, including against SARS-CoV-2 variants of concern. BNT162b2 boosting was associated with a rapid increase of Omicron neutralizing antibodies that peaked at a median (IQR) titer of 1018 (699-1646) at week 2 and declined by 6.9-fold to a median (IQR) titer of 148 (95-266) by week 16. Ad26.COV2.S boosting was associated with increased Omicron neutralizing antibodies titers that peaked at a median (IQR) of 859 (467-1838) week 4 and declined by 2.1-fold to a median (IQR) of 403 (208-1130) by week 16. Conclusions and Relevance: Heterologous Ad26.COV2.S boosting was associated with durable humoral and cellular immune responses in individuals who originally received the BNT162b2 vaccine. These data suggest potential benefits of heterologous prime-boost vaccine regimens for SARS-CoV-2.

Reduced SARS-CoV-2 disease outcomes in Syrian hamsters receiving immune sera: Quantitative image analysis in pathologic assessments.

There is a need to standardize pathologic endpoints in animal models of SARS-CoV-2 infection to help benchmark study quality, improve cross-institutional comparison of data, and assess therapeutic efficacy so that potential drugs and vaccines for SARS-CoV-2 can rapidly advance. The Syrian hamster model is a tractable small animal model for COVID-19 that models clinical disease in humans. Using the hamster model, the authors used traditional pathologic assessment with quantitative image analysis to assess disease outcomes in hamsters administered polyclonal immune sera from previously challenged rhesus macaques. The authors then used quantitative image analysis to assess pathologic endpoints across studies performed at different institutions using different tissue processing protocols. The authors detail pathological features of SARS-CoV-2 infection longitudinally and use immunohistochemistry to quantify myeloid cells and T lymphocyte infiltrates during SARS-CoV-2 infection. High-dose immune sera protected hamsters from weight loss and diminished viral replication in tissues and reduced lung lesions. Cumulative pathology scoring correlated with weight loss and was robust in distinguishing IgG efficacy. In formalin-infused lungs, quantitative measurement of percent area affected also correlated with weight loss but was less robust in non-formalin-infused lungs. Longitudinal immunohistochemical assessment of interstitial macrophage infiltrates showed that peak infiltration corresponded to weight loss, yet quantitative assessment of macrophage, neutrophil, and CD3+ T lymphocyte numbers did not distinguish IgG treatment effects. Here, the authors show that quantitative image analysis was a useful adjunct tool for assessing SARS-CoV-2 treatment outcomes in the hamster model.

Durability and expansion of neutralizing antibody breadth following Ad26.COV2.S vaccination of mice.

Emerging SARS-CoV-2 variants with the potential to escape binding and neutralizing antibody responses pose a threat to vaccine efficacy. We recently reported expansion of broadly neutralizing activity of vaccine-elicited antibodies in humans 8 months following a single immunization with Ad26.COV2.S. Here, we assessed the 15-month durability of antibody responses and their neutralizing capacity to B.1.617.2 (delta) and B.1.351 (beta) variants following a single immunization of Ad26.COV2.S in mice. We report the persistence of binding and neutralizing antibody titers following immunization with a concomitant increase in neutralizing antibody breadth to delta and beta variants over time. Evaluation of bone marrow and spleen at 15 months postimmunization revealed that Ad26.COV2.S-immunized mice tissues contained spike-specific antibody-secreting cells. We conclude that immunization with Ad26.COV2.S elicits a robust immune response against SARS-CoV-2 spike, which expands over time to neutralize delta and beta variants more robustly, and seeds bone marrow and spleen with long-lived spike-specific antibody-secreting cells. These data extend previous findings in humans and support the use of a mouse model as a potential tool to further explore the dynamics of the humoral immune response following vaccination with Ad26.COV2.S.

A homologous or variant booster vaccine after Ad26.COV2.S immunization enhances SARS-CoV-2-specific immune responses in rhesus macaques.

Ad26.COV2.S has demonstrated durability and clinical efficacy against symptomatic COVID-19 in humans. In this study, we report the correlates of durability of humoral and cellular immune responses in 20 rhesus macaques immunized with single-shot Ad26.COV2.S and the immunogenicity of a booster shot at 8 to 10 months after the initial immunization. Ad26.COV2.S elicited durable binding and neutralizing antibodies as well as memory B cells and long-lived bone marrow plasma cells. Innate immune responses and bone marrow plasma cell responses correlated with durable antibody responses. After Ad26.COV2.S boost immunization, binding and neutralizing antibody responses against multiple SARS-CoV-2 variants increased 31- to 69-fold and 23- to 43-fold, respectively, compared with preboost concentrations. Antigen-specific B cell and T cell responses also increased substantially after the boost immunization. Boosting with a modified Ad26.COV2.S.351 vaccine expressing the SARS-CoV-2 spike protein from the beta variant led to largely comparable responses with slightly higher beta- and omicron-specific humoral immune responses. These data demonstrate that a late boost with Ad26.COV2.S or Ad26.COV2.S.351 resulted in a marked increase in humoral and cellular immune responses that were highly cross-reactive across multiple SARS-CoV-2 variants in rhesus macaques.

Immunity elicited by natural infection or Ad26.COV2.S vaccination protects hamsters against SARS-CoV-2 variants of concern.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern have emerged and may pose a threat to both the efficacy of vaccines based on the original WA1/2020 strain and the natural immunity induced by infection with earlier SARS-CoV-2 variants. We investigated how mutations in the spike protein of circulating SARS-CoV-2 variants, which have been shown to partially evade neutralizing antibodies, affect natural and vaccine-induced immunity. We adapted a Syrian hamster model of moderate to severe clinical disease for two variant strains of SARS-CoV-2: B.1.1.7 (alpha variant) and B.1.351 (beta variant). We then assessed the protective efficacy conferred by either natural immunity from WA1/2020 infection or by vaccination with a single dose of the adenovirus serotype 26 vaccine, Ad26.COV2.S. Primary infection with the WA1/2020 strain provided potent protection against weight loss and viral replication in lungs after rechallenge with WA1/2020, B.1.1.7, or B.1.351. Ad26.COV2.S induced cross-reactive binding and neutralizing antibodies that were reduced against the B.1.351 strain compared with WA1/2020 but nevertheless still provided robust protection against B.1.351 challenge, as measured by weight loss and pathology scoring in the lungs. Together, these data support hamsters as a preclinical model to study protection against emerging variants of SARS-CoV-2 conferred by prior infection or vaccination.

Protective efficacy of Ad26.COV2.S against SARS-CoV-2 B.1.351 in macaques.

The emergence of SARS-CoV-2 variants that partially evade neutralizing antibodies poses a threat to the efficacy of current COVID-19 vaccines1,2. The Ad26.COV2.S vaccine expresses a stabilized spike protein from the WA1/2020 strain of SARS-CoV-2, and has recently demonstrated protective efficacy against symptomatic COVID-19 in humans in several geographical regions-including in South Africa, where 95% of sequenced viruses in cases of COVID-19 were the B.1.351 variant3. Here we show that Ad26.COV2.S elicits humoral and cellular immune responses that cross-react with the B.1.351 variant and protects against B.1.351 challenge in rhesus macaques. Ad26.COV2.S induced lower binding and neutralizing antibodies against B.1.351 as compared to WA1/2020, but elicited comparable CD8 and CD4 T cell responses against the WA1/2020, B.1.351, B.1.1.7, P.1 and CAL.20C variants. B.1.351 infection of control rhesus macaques resulted in higher levels of virus replication in bronchoalveolar lavage and nasal swabs than did WA1/2020 infection. Ad26.COV2.S provided robust protection against both WA1/2020 and B.1.351, although we observed higher levels of virus in vaccinated macaques after B.1.351 challenge. These data demonstrate that Ad26.COV2.S provided robust protection against B.1.351 challenge in rhesus macaques. Our findings have important implications for vaccine control of SARS-CoV-2 variants of concern.

Immunogenicity of COVID-19 mRNA Vaccines in Pregnant and Lactating Women.

Importance: Pregnant women are at increased risk of morbidity and mortality from COVID-19 but have been excluded from the phase 3 COVID-19 vaccine trials. Data on vaccine safety and immunogenicity in these populations are therefore limited. Objective: To evaluate the immunogenicity of COVID-19 messenger RNA (mRNA) vaccines in pregnant and lactating women, including against emerging SARS-CoV-2 variants of concern. Design, Setting, and Participants: An exploratory, descriptive, prospective cohort study enrolled 103 women who received a COVID-19 vaccine from December 2020 through March 2021 and 28 women who had confirmed SARS-CoV-2 infection from April 2020 through March 2021 (the last follow-up date was March 26, 2021). This study enrolled 30 pregnant, 16 lactating, and 57 neither pregnant nor lactating women who received either the mRNA-1273 (Moderna) or BNT162b2 (Pfizer-BioNTech) COVID-19 vaccines and 22 pregnant and 6 nonpregnant unvaccinated women with SARS-CoV-2 infection. Main Outcomes and Measures: SARS-CoV-2 receptor binding domain binding, neutralizing, and functional nonneutralizing antibody responses from pregnant, lactating, and nonpregnant women were assessed following vaccination. Spike-specific T-cell responses were evaluated using IFN-γ enzyme-linked immunospot and multiparameter intracellular cytokine-staining assays. Humoral and cellular immune responses were determined against the original SARS-CoV-2 USA-WA1/2020 strain as well as against the B.1.1.7 and B.1.351 variants. Results: This study enrolled 103 women aged 18 to 45 years (66% non-Hispanic White) who received a COVID-19 mRNA vaccine. After the second vaccine dose, fever was reported in 4 pregnant women (14%; SD, 6%), 7 lactating women (44%; SD, 12%), and 27 nonpregnant women (52%; SD, 7%). Binding, neutralizing, and functional nonneutralizing antibody responses as well as CD4 and CD8 T-cell responses were present in pregnant, lactating, and nonpregnant women following vaccination. Binding and neutralizing antibodies were also observed in infant cord blood and breast milk. Binding and neutralizing antibody titers against the SARS-CoV-2 B.1.1.7 and B.1.351 variants of concern were reduced, but T-cell responses were preserved against viral variants. Conclusion and Relevance: In this exploratory analysis of a convenience sample, receipt of a COVID-19 mRNA vaccine was immunogenic in pregnant women, and vaccine-elicited antibodies were transported to infant cord blood and breast milk. Pregnant and nonpregnant women who were vaccinated developed cross-reactive antibody responses and T-cell responses against SARS-CoV-2 variants of concern.

Persistence of viral RNA in lymph nodes in ART-suppressed SIV/SHIV-infected Rhesus Macaques.

The establishment of a long-lived viral reservoir is the key obstacle for achieving an HIV-1 cure. However, the anatomic, virologic, and immunologic features of the viral reservoir in tissues during antiretroviral therapy (ART) remain poorly understood. Here we present a comprehensive necroscopic analysis of the SIV/SHIV viral reservoir in multiple lymphoid and non-lymphoid tissues from SIV/SHIV-infected rhesus macaques suppressed with ART for one year. Viral DNA is observed broadly in multiple tissues and is comparable in animals that had initiated ART at week 1 or week 52 of infection. In contrast, viral RNA is restricted primarily to lymph nodes. Ongoing viral RNA transcription is not the result of unsuppressed viral replication, as single-genome amplification and subsequent phylogenetic analysis do not show evidence of viral evolution. Gag-specific CD8+ T cell responses are predominantly observed in secondary lymphoid organs in animals chronically infected prior to ART and these responses are dominated by CD69+ populations. Overall, we observe that the viral reservoir in rhesus macaques is widely distributed across multiple tissue sites and that lymphoid tissues act as a site of persistent viral RNA transcription under conditions of long-term ART suppression.

Deletion of the SARS-CoV-2 Spike Cytoplasmic Tail Increases Infectivity in Pseudovirus Neutralization Assays.

Pseudotyped viruses are valuable tools for studying virulent or lethal viral pathogens that need to be handled in biosafety level 3 (BSL-3) or higher facilities. With the explosive spread of the coronavirus disease 2019 (COVID-19) pandemic, the establishment of a BSL-2 adapted SARS-CoV-2 pseudovirus neutralization assay is needed to facilitate the development of countermeasures. Here we describe an approach to generate a single-round lentiviral vector-based SARS-CoV-2 pseudovirus, which produced a signal more than 2 logs above background. Specifically, a SARS-CoV-2 spike variant with a cytoplasmic tail deletion of 13 amino acids, termed SΔCT13, conferred enhanced spike incorporation into pseudovirions and increased viral entry into cells as compared with full-length spike (S). We further compared S and SΔCT13 in terms of their sensitivity to vaccine sera, purified convalescent IgG, hACE2-mIgG, and the virus entry inhibitor BafA1. We developed a SΔCT13-based pseudovirus neutralization assay and defined key assay characteristics, including linearity, limit of detection, and intra- and intermediate-assay precision. Our data demonstrate that the SΔCT13-based pseudovirus shows enhanced infectivity in target cells, which will facilitate the assessment of humoral immunity to SARS-CoV-2 infection, antibody therapeutics, and vaccination. This pseudovirus neutralization assay can also be readily adapted to SARS-CoV-2 variants that emerge.IMPORTANCESARS-CoV-2 is the etiologic agent of the COVID-19 pandemic. The development of a high throughput pseudovirus neutralization assay is critical for the development of vaccines and immune-based therapeutics. In this study, we show that deletion of the cytoplasmic tail of the SARS-CoV-2 spike leads to pseudoviruses with enhanced infectivity. This SΔCT13-based pseudovirus neutralization assay should be broadly useful for the field.

Adenovirus-vectored vaccine containing multidimensionally conserved parts of the HIV proteome is immunogenic in rhesus macaques.

An effective vaccine that can protect against HIV infection does not exist. A major reason why a vaccine is not available is the high mutability of the virus, which enables it to evolve mutations that can evade human immune responses. This challenge is exacerbated by the ability of the virus to evolve compensatory mutations that can partially restore the fitness cost of immune-evading mutations. Based on the fitness landscapes of HIV proteins that account for the effects of coupled mutations, we designed a single long peptide immunogen comprising parts of the HIV proteome wherein mutations are likely to be deleterious regardless of the sequence of the rest of the viral protein. This immunogen was then stably expressed in adenovirus vectors that are currently in clinical development. Macaques immunized with these vaccine constructs exhibited T-cell responses that were comparable in magnitude to animals immunized with adenovirus vectors with whole HIV protein inserts. Moreover, the T-cell responses in immunized macaques strongly targeted regions contained in our immunogen. These results suggest that further studies aimed toward using our vaccine construct for HIV prophylaxis and cure are warranted.

Correlates of protection against SARS-CoV-2 in rhesus macaques.

Recent studies have reported the protective efficacy of both natural1 and vaccine-induced2-7 immunity against challenge with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in rhesus macaques. However, the importance of humoral and cellular immunity for protection against infection with SARS-CoV-2 remains to be determined. Here we show that the adoptive transfer of purified IgG from convalescent rhesus macaques (Macaca mulatta) protects naive recipient macaques against challenge with SARS-CoV-2 in a dose-dependent fashion. Depletion of CD8+ T cells in convalescent macaques partially abrogated the protective efficacy of natural immunity against rechallenge with SARS-CoV-2, which suggests a role for cellular immunity in the context of waning or subprotective antibody titres. These data demonstrate that relatively low antibody titres are sufficient for protection against SARS-CoV-2 in rhesus macaques, and that cellular immune responses may contribute to protection if antibody responses are suboptimal. We also show that higher antibody titres are required for treatment of SARS-CoV-2 infection in macaques. These findings have implications for the development of SARS-CoV-2 vaccines and immune-based therapeutic agents.

Passive Transfer of Vaccine-Elicited Antibodies Protects against SIV in Rhesus Macaques.

Several HIV-1 and SIV vaccine candidates have shown partial protection against viral challenges in rhesus macaques. However, the protective efficacy of vaccine-elicited polyclonal antibodies has not previously been demonstrated in adoptive transfer studies in nonhuman primates. In this study, we show that passive transfer of purified antibodies from vaccinated macaques can protect naive animals against SIVmac251 challenges. We vaccinated 30 rhesus macaques with Ad26-SIV Env/Gag/Pol and SIV Env gp140 protein vaccines and assessed the induction of antibody responses and a putative protective signature. This signature included multiple antibody functions and correlated with upregulation of interferon pathways in vaccinated animals. Adoptive transfer of purified immunoglobulin G (IgG) from the vaccinated animals with the most robust protective signatures provided partial protection against SIVmac251 challenges in naive recipient rhesus macaques. These data demonstrate the protective efficacy of purified vaccine-elicited antiviral antibodies in this model, even in the absence of virus neutralization.