Antibodies to S2 domain of SARS-CoV-2 spike protein in Moderna mRNA vaccinated subjects sustain antibody-dependent NK cell-mediated cell cytotoxicity against Omicron BA.1.

Vaccination with the primary two-dose series of SARS-CoV-2 mRNA protects against infection with the ancestral strain, and limits the presentation of severe disease after re-infection by multiple variants of concern (VOC), including Omicron, despite the lack of a strong neutralizing response to these variants. We compared antibody responses in serum samples collected from mRNA-1273 (Moderna) vaccinated subjects to identify mechanisms of immune escape and cross-protection. Using pseudovirus constructs containing domain-specific amino acid changes representative of Omicron BA.1, combined with domain competition and RBD-antibody depletion, we showed that RBD antibodies were primarily responsible for virus neutralization and variant escape. Antibodies to NTD played a less significant role in antibody neutralization but acted along with RBD to enhance neutralization. S2 of Omicron BA.1 had no impact on neutralization escape, suggesting it is a less critical domain for antibody neutralization; however, it was as capable as S1 at eliciting IgG3 responses and NK-cell mediated, antibody-dependent cell cytotoxicity (ADCC). Antibody neutralization and ADCC activities to RBD, NTD, and S1 were all prone to BA.1 escape. In contrast, ADCC activities to S2 resisted BA.1 escape. In conclusion, S2 antibodies showed potent ADCC function and resisted Omicron BA.1 escape, suggesting that S2 contributes to cross-protection against Omicron BA.1. In line with its conserved nature, S2 may hold promise as a vaccine target against future variants of SARS-CoV-2.

Antibody Responses to the SARS-CoV-2 Ancestral Strain and Omicron Variants in Moderna mRNA-1273 Vaccinated Active-Duty US Navy Sailors and Marines.

Omicron and its subvariants have steadily gained greater capability of immune escape compared to other variants of concern, resulting in an increased incidence of reinfections even among vaccinated individuals. We evaluated the antibody response to Omicron BA.1, BA.2, and BA.4/5 in US military members vaccinated with the primary 2-dose series of Moderna mRNA-1273 in a cross-sectional study. While nearly all vaccinated participants had sustained spike (S) IgG and neutralizing antibodies (ND50) to the ancestral strain, only 7.7% participants had detectable ND50 to Omicron BA.1 at 8 months postvaccination. The neutralizing antibody response to BA.2 and BA.5 was similarly reduced. The reduced antibody neutralization of Omicron correlated with the decreased antibody binding to the receptor-binding domain. The participants' seropositivity to the nuclear protein positively correlated with ND50. Our data emphasizes the need for continuous vigilance in monitoring for emerging variants and the need to identify potential alternative targets for vaccine design.

Serum Fc-Mediated Monocyte Phagocytosis Activity Is Stable for Several Months after SARS-CoV-2 Asymptomatic and Mildly Symptomatic Infection.

We investigated the temporal profile of multiple components of the serological response after asymptomatic or mildly symptomatic SARS-CoV-2 infection, in a cohort of 67 previously SARS-CoV-2 naive young adults, up to 8.5 months after infection. We found a significant decrease of spike IgG and neutralization antibody titers from early (11 to 56 days) to late (4 to 8.5 months) time points postinfection. Over the study period, S1-specific IgG levels declined significantly faster than that of the S2-specific IgG. Further, serum antibodies from PCR-confirmed participants cross-recognized S2, but not S1, of the betacoronaviruses HKU1 and OC43, suggesting a greater degree of cross-reactivity of S2 among betacoronaviruses. Antibody-Dependent Natural Killer cell Activation (ADNKA) was detected at the early time point but significantly decreased at the late time point. Induction of serum Antibody-Dependent Monocyte Phagocytosis (ADMP) was detected in all the infected participants, and its levels remained stable over time. Additionally, a reduced percentage of participants had detectable neutralizing activity against the Beta (50%), Gamma (61 to 67%), and Delta (90 to 94%) variants, both early and late postinfection, compared to the ancestral strain (100%). Antibody binding to S1 and RBD of Beta, Gamma, Delta (1.7 to 2.3-fold decrease), and Omicron (10 to 16-fold decrease) variants was also significantly reduced compared to the ancestral SARS-CoV-2 strain. Overall, we found variable temporal profiles of specific components and functionality of the serological response to SARS-CoV-2 in young adults, which is characterized by lasting, but decreased, neutralizing activity and antibody binding to S1, stable ADMP activity, and relatively stable S2-specific IgG levels. IMPORTANCE Adaptive immunity mediated by antibodies is important for controlling SARS-CoV-2 infection. While vaccines against COVID-19 are currently widely distributed, a high proportion of the global population is still unvaccinated. Therefore, understanding the dynamics and maintenance of the naive humoral immune response to SARS-CoV-2 is of great importance. In addition, long-term responses after asymptomatic infection are not well-characterized, given the challenges in identifying such cases. Here, we investigated the longitudinal humoral profile in a well-characterized cohort of young adults with documented asymptomatic or mildly symptomatic SARS-CoV-2 infection. By analyzing samples collected preinfection, early after infection and during late convalescence, we found that, while neutralizing activity decreased over time, high levels of serum S2 IgG and Antibody-Dependent Monocyte Phagocytosis (ADMP) activity were maintained up to 8.5 months after infection. This suggests that a subset of antibodies with specific functions could contribute to long-term protection against SARS-CoV-2 in convalescent unvaccinated individuals.

Asymptomatic or symptomatic SARS-CoV-2 infection plus vaccination confers increased adaptive immunity to variants of concern.

The ongoing evolution of SARS-CoV-2 requires monitoring the capability of immune responses to cross-recognize Variants of Concern (VOC). In this cross-sectional study, we examined serological and cell-mediated immune memory to SARS-CoV-2 variants, including Omicron, among a cohort of 18-21-year-old Marines with a history of either asymptomatic or mild SARS-CoV-2 infection 6 to 14 months earlier. Among the 210 participants in the study, 169 were unvaccinated while 41 received 2 doses of mRNA-based COVID-19 vaccines. Vaccination of previously infected participants strongly boosted neutralizing and binding activity and memory B and T cell responses including the recognition of Omicron, compared to infected but unvaccinated participants. Additionally, no measurable differences were observed in immune memory in healthy young adults with previous symptomatic or asymptomatic infections, for ancestral or variant strains. These results provide mechanistic immunological insights into population-based differences observed in immunity against Omicron and other variants among individuals with different clinical histories.

Memory B-Cell Development After Asymptomatic or Mild Symptomatic SARS-CoV-2 Infection.

BACKGROUND: The development of memory B cells after asymptomatic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is not well understood. METHODS: We compared spike antibody titers, pseudovirus neutralizing antibody titers, and memory B-cell responses among SARS-CoV-2 PCR-positive Marine recruits who either reported asymptomatic or symptomatic infection. RESULTS: Thirty-six asymptomatic participants exhibited similar spike IgG titers, spike IgA titers, and pseudovirus neutralization titers compared to 30 symptomatic participants. Pseudovirus neutralization and spike IgG titers showed significant positive correlations with frequency of memory B cells. CONCLUSIONS: Among young adults, asymptomatic SARS-CoV-2 infection induced antibody and memory B-cell responses comparable to mild symptomatic infection.

Enhanced Immunogenicity of Inactivated Dengue Vaccines by Novel Polysaccharide-Based Adjuvants in Mice.

Dengue fever, caused by any of four dengue viruses (DENV1-4), is a major global burden. Currently, there is no effective vaccine that prevents infection in dengue naïve populations. We tested the ability of two novel adjuvants (Advax-PEI and Advax-2), using aluminum hydroxide (alum) as control, to enhance the immunogenicity of formalin- or psoralen-inactivated (PIV or PsIV) DENV2 vaccines in mice. Mice were vaccinated on days 0 and 30, and serum samples were collected on days 30, 60, 90, and 101. Neutralizing antibodies were determined by microneutralization (MN) assays, and the geometric mean 50% MN (MN50) titers were calculated. For the PIV groups, after one dose MN50 titers were higher in the novel adjuvant groups compared to the alum control, while MN50 titers were comparable between the adjuvant groups after the second dose. For the PsIV groups, both novel adjuvants induced higher MN50 titers than the alum control after the second dose. Spleen cells were collected on days 45 and 101 for enzyme-linked immunospot (ELISPOT) for IFNγ and IL4. Both PIV and PsIV groups elicited different degrees of IFNγ and IL4 responses. Overall, Advax-2 gave the best responses just ahead of Advax-PEI. Given Advax-2's extensive human experience in other vaccine applications, it will be pursued for further development.

Pseudovirus-Based Assays for the Measurement of Antibody-Mediated Neutralization of SARS-CoV-2.

SARS-CoV-2 has emerged as a significant cause of morbidity and mortality worldwide. Virus neutralization assays are critical for the development and evaluation of vaccines and immunotherapeutics, as well as for conducting basic research into the immune response, spread, and pathogenesis of this disease. However, neutralization assays traditionally require the use of infectious virus which must be carefully handled in a BSL-3 setting, thus complicating the assay and restricting its use to labs with access to BSL-3 facilities. Pseudovirus-based assays are an alternative to the use of infectious virus. SARS-CoV-2 pseudovirus contains only the spike structural protein, and infection results in a single round of replication, thus allowing for the assay to be run safely under BSL-2 conditions. In this chapter, we describe protocols and considerations for the production and titration of lentivirus-based SARS-CoV-2 pseudovirus, as well as for running and analysis of FACS-based pseudovirus neutralization assays.

CHARM: COVID-19 Health Action Response for Marines-Association of antigen-specific interferon-gamma and IL2 responses with asymptomatic and symptomatic infections after a positive qPCR SARS-CoV-2 test.

SARS-CoV-2 T cell responses are associated with COVID-19 recovery, and Class I- and Class II-restricted epitopes have been identified in the spike (S), nucleocapsid (N) and membrane (M) proteins and others. This prospective COVID-19 Health Action Response for Marines (CHARM) study enabled assessment of T cell responses against S, N and M proteins in symptomatic and asymptomatic SARS-CoV-2 infected participants. At enrollment all participants were negative by qPCR; follow-up occurred biweekly and bimonthly for the next 6 weeks. Study participants who tested positive by qPCR SARS-CoV-2 test were enrolled in an immune response sub-study. FluoroSpot interferon-gamma (IFN-γ) and IL2 responses following qPCR-confirmed infection at enrollment (day 0), day 7 and 14 and more than 28 days later were measured using pools of 17mer peptides covering S, N, and M proteins, or CD4+CD8 peptide pools containing predicted epitopes from multiple SARS-CoV-2 antigens. Among 124 asymptomatic and 105 symptomatic participants, SARS-CoV-2 infection generated IFN-γ responses to the S, N and M proteins that persisted longer in asymptomatic cases. IFN-γ responses were significantly (p = 0.001) more frequent to the N pool (51.4%) than the M pool (18.9%) among asymptomatic but not symptomatic subjects. Asymptomatic IFN-γ responders to the CD4+CD8 pool responded more frequently to the S pool (55.6%) and N pool (57.1%), than the M pool (7.1%), but not symptomatic participants. The frequencies of IFN-γ responses to the S and N+M pools peaked 7 days after the positive qPCR test among asymptomatic (S pool: 22.2%; N+M pool: 28.7%) and symptomatic (S pool: 15.3%; N+M pool 21.9%) participants and dropped by >28 days. Magnitudes of post-infection IFN-γ and IL2 responses to the N+M pool were significantly correlated with IFN-γ and IL2 responses to the N and M pools. These data further support the central role of Th1-biased cell mediated immunity IFN-γ and IL2 responses, particularly to the N protein, in controlling COVID-19 symptoms, and justify T cell-based COVID-19 vaccines that include the N and S proteins.

T cell and memory B cell responses in tetravalent DNA, tetravalent inactivated and tetravalent live-attenuated prime-boost dengue vaccines in rhesus macaques.

We previously reported the efficacy of prime-boost vaccination using three tetravalent (T) dengue vaccines, DNA (TDNA), purified inactivated vaccine (TPIV), and live attenuated vaccine (TLAV). We demonstrated that the TPIV/TLAV prime-boost vaccination yielded the highest and most durable neutralizing antibodies and 100% protection to all 4 serotypes of dengue virus in rhesus macaques. This study compares gene transcription, T and B cell responses elicited by these prime-boost combinations in rhesus macaques. This study shows that the TLAV vaccine increased the expression of the innate immune genes, DDX58 and TLR7, IL1A, IL1B, TNF, CXCL8, CXCL10, IRF1, IRF7, and IFNB, more robustly as compared to TDNA and TPIV vaccines. Overall, two doses of TDNA and one dose of TLAV efficiently elicited a T cell IFNγ response to PrM/E with a comparable magnitude. Compared to TDNA vaccine, the TLAV vaccine elicited additional IFNγ response to C, NS1, NS3, and NS5. The TPIV vaccine alone produced poor IFNγ response; however, the TLAV significantly boosted its IFNγ response. The T cell response repertoire associated with TPIV/TLAV prime-boost was to both the structural C/PrM/E and NS proteins, and the T cells were multifunctional as the CD4+ T cells produced IFNγ, TNF α, and IL2 and the CD8+ cells produced TNF α and IFNγ. Opposite to the pattern of CMI, the TPIV vaccine alone elicited the highest BMem compared to the other two vaccines, which continuously remained as the highest after boosting. In summary, the TDNA and TLAV vaccines elicited a strong T cell response whereas the TPIV vaccine elicited a superior BMem. The T cell response of the TPIV vaccine was significantly boosted by the TLAV vaccine. The elevated T cell response may have provided T cell help for a sustained antibody response for TPIV/TLAV vaccines, which is required for a protective immunity against a live virus challenge.

Immunogenicity of Adjuvanted Psoralen-Inactivated SARS-CoV-2 Vaccines and SARS-CoV-2 Spike Protein DNA Vaccines in BALB/c Mice.

The development of a safe and effective vaccine to protect against COVID-19 is a global priority due to the current high SARS-CoV-2 infection rate. Currently, there are over 160 SARS-CoV-2 vaccine candidates at the clinical or pre-clinical stages of development. Of these, there are only three whole-virus vaccine candidates produced using ß-propiolactone or formalin inactivation. Here, we prepared a whole-virus SARS-CoV-2 vaccine (SARS-CoV-2 PsIV) using a novel psoralen inactivation method and evaluated its immunogenicity in mice using two different adjuvants, alum and Advax-2. We compared the immunogenicity of SARS-CoV-2 PsIV against SARS-CoV-2 DNA vaccines expressing either full-length or truncated spike proteins. We also compared the psoralen-inactivated vaccine against a DNA prime, psoralen-inactivated vaccine boost regimen. After two doses, the psoralen-inactivated vaccine, when administered with alum or Advax-2 adjuvants, generated a dose-dependent neutralizing antibody responses in mice. Overall, the pattern of cytokine ELISPOT responses to antigen-stimulation observed in this study indicates that SARS-CoV-2 PsIV with the alum adjuvant promotes a Th2-type response, while SARS-CoV-2 PsIV with the Advax-2 adjuvant promotes a Th1-type response.

Distribution of Capsular Types of Campylobacter jejuni Isolates from Symptomatic and Asymptomatic Children in Peru.

Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide. A capsular polysaccharide (CPS) conjugate vaccine is under development and requires determination of the valency. However, distribution of CPS types circulating globally is presently poorly described. We aimed to determine whether CPS type distribution in Peru differs from that in other endemic regions. We used a multiplex polymerase chain reaction (PCR) assay for the detection of CPS encoding genes capable of distinguishing all 35 CPS types on Campylobacter isolates in two prospective communities based studies conducted in cohorts of children less than 59 months of age in Peru. Results showed that CPS type HS4 complex was the most prevalent, followed by HS3 complex and HS15. Differences in CPS type for symptomatology were not statistically significant. Most subjects demonstrated repeated infections over time with different CPS types, suggesting that CPS types may confer of a level of homologous protective immunity. In this dataset, some differences in CPS type distribution were observed in comparison to other low-middle income countries. Further studies need to be conducted in endemic areas to increase our knowledge of CPS type distribution and guide vaccine development.

NK Cells Activated through Antibody-Dependent Cell Cytotoxicity and Armed with Degranulation/IFN-γ Production Suppress Antibody-dependent Enhancement of Dengue Viral Infection.

Antibody (Ab)-dependent enhancement (ADE) is a hypothesized mechanism of increased disease severity during secondary dengue virus (DENV) infection. This study investigates Ab-dependent cell cytotoxicity (ADCC) in counteracting ADE. In our system, DENV and DENV-immune sera were added to peripheral blood mononuclear cells (PBMCs), and ADE and NK cell activation were simultaneously monitored. ADE was detected in monocytes and a concurrent activation of NK cells was observed. Activated NK cells expressed IFN-γ and CD107a. IFN-γ was detected at 24 hours (24 h) followed by a rapid decline; CD107a expression peaked at 48 h and persisted for >7 days. Optimal activation of NK cells required the presence of enhancement serum together with ADE-affected monocytes and soluble factors, suggesting the coexistence of the counteractive ADCC Abs, in the same ADE-serum, capable of strongly promoting NK cell activation. The function of NK cells against ADE was demonstrated using a depletion assay. NK cell-depleted PBMCs had increased ADE as compared to whole PBMCs. Conversely, adding activated NK cells back into the NK-depleted-PBMCs or to purified monocytes decreased ADE. Blocking IFN-γ expression also increased ADE. The study suggests that under ADE conditions, NK cells can be activated by ADCC Abs and can control the magnitude of ADE.