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Background Genogroup II noroviruses are the most common cause of acute infectious gastroenteritis. We evaluated the use of a new GII.2 inoculum in a human challenge. Methods Forty-four healthy adults (36 secretor-positive and 8 secretor-negative for histo-blood group antigens) were challenged with ascending doses of a new safety-tested Snow Mountain virus (SMV) GII.2 norovirus inoculum (1.2 × 104 to 1.2 × 107 genome equivalent copies [GEC]; n = 38) or placebo (n = 6). Illness was defined as diarrhea and/or vomiting postchallenge in subjects with evidence of infection (defined as GII.2 norovirus RNA detection in stool and/or anti-SMV immunoglobulin G [IgG] seroconversion). Results The highest dose was associated with SMV infection in 90%, and illness in 70% of subjects with 10 of 12 secretor-positive (83%) and 4 of 8 secretor-negative (50%) becoming ill. There was no association between prechallenge anti-SMV serum IgG concentration, carbohydrate-binding blockade antibody, or salivary immunoglobulin A and infection. The median infectious dose (ID50) was 5.1 × 105 GEC. Conclusions High rates of infection and illness were observed in both secretor-positive and secretor-negative subjects in this challenge study. However, a high dose will be required to achieve the target of 75% illness to make this an efficient model for evaluating potential norovirus vaccines and therapeutics. Clinical Trials Registration NCT02473224.

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 - 100 μg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naïve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.mRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

Neutralizing antibody responses gradually wane after vaccination with mRNA-1273 against several variants of concern (VOC), and additional boost vaccinations may be required to sustain immunity and protection. Here, we evaluated the immune responses in nonhuman primates that received 100 µg of mRNA-1273 vaccine at 0 and 4 weeks and were boosted at week 29 with mRNA-1273 (homologous) or mRNA-1273.β (heterologous), which encompasses the spike sequence of the B.1.351 (beta or β) variant. Reciprocal ID50pseudovirus neutralizing antibody geometric mean titers (GMT) against live SARS-CoV-2 D614G and the β variant, were 4700 and 765, respectively, at week 6, the peak of primary response, and 644 and 553, respectively, at a 5-month post-vaccination memory time point. Two weeks following homologous or heterologous boost β-specific reciprocal ID50GMT were 5000 and 3000, respectively. At week 38, animals were challenged in the upper and lower airway with the β variant. Two days post-challenge, viral replication was low to undetectable in both BAL and nasal swabs in most of the boosted animals. These data show that boosting with the homologous mRNA-1273 vaccine six months after primary immunization provides up to a 20-fold increase in neutralizing antibody responses across all VOC, which may be required to sustain high-level protection against severe disease, especially for at-risk populations.One-sentence summarymRNA-1273 boosted nonhuman primates have increased immune responses and are protected against SARS-CoV-2 beta infection.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike protein trimers (preS dTM) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHPs). Binding and functional neutralization assays and systems serology revealed that the vaccinated NHP developed AS03-dependent multifunctional humoral responses that targeted distinct domains of the spike protein and bound to a variety of Fc receptors mediating immune cell effector functions in vitro. The neutralizing 50% inhibitory concentration titers for pseudovirus and live SARS-CoV-2 were higher than titers for a panel of human convalescent serum samples. NHPs were challenged intranasally and intratracheally with a high dose (3 × 106 plaque forming units) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days after challenge, vaccinated NHPs showed rapid control of viral replication in both the upper and lower airways. Vaccinated NHPs also had increased spike protein-specific immunoglobulin G (IgG) antibody responses in the lung as early as 2 days after challenge. Moreover, passive transfer of vaccine-induced IgG to hamsters mediated protection from subsequent SARS-CoV-2 challenge. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine were sufficient to mediate protection against SARS-CoV-2 in NHPs and that rapid anamnestic antibody responses in the lung may be a key mechanism for protection.

Unadjuvanted A/H7N9 vaccines are poorly immunogenic. The immune response is improved with the addition of MF59, an oil-in-water adjuvant. However, the cellular immunologic responses of MF59-adjuvanted A/H7N9 vaccine are not fully understood.37 participants were vaccinated with 2 doses of 2013 influenza A/H7N9 vaccine (at Days 1 and 21) with or without MF59 and enrolled in an immunology substudy. Responses were assessed at multiple timepoints (Days 0, 8, 21, 29, and 42) for hemagglutination inhibition (HAI) and neutralizing antibody (Neut) assays, memory B cell responses by enzyme-linked ImmunoSpot; circulating follicular helper T cells (cTMF59-adjuvanted influenza A/H7N9 vaccine induced significantly higher hemagglutination inhibition (HAI) and neutralizing antibody (Neut) responses when compared to unadjuvanted vaccine. The adjuvanted vaccine elicited significantly higher levels of Inducible T-cell Co-Stimulator (ICOS) expression by CXCR3cT

Adjuvants enhance the magnitude and the durability of the immune response to vaccines. However, there is a paucity of comparative studies on the nature of the immune responses stimulated by leading adjuvant candidates. In this study, we compared five clinically relevant adjuvants in mice—alum, AS03 (a squalene-based adjuvant supplemented with α-tocopherol), AS37 (a TLR7 ligand emulsified in alum), CpG1018 (a TLR9 ligand emulsified in alum), O/W 1849101 (a squalene-based adjuvant)—for their capacity to stimulate immune responses when combined with a subunit vaccine under clinical development. We found that all four of the adjuvant candidates surpassed alum with respect to their capacity to induce enhanced and durable antigen-specific antibody responses. The TLR-agonist-based adjuvants CpG1018 (TLR9) and AS37 (TLR7) induced Th1-skewed CD4+ T cell responses, while alum, O/W, and AS03 induced a balanced Th1/Th2 response. Consistent with this, adjuvants induced distinct patterns of early innate responses. Finally, vaccines adjuvanted with AS03, AS37, and CpG1018/alum-induced durable neutralizing-antibody responses and significant protection against the B.1.351 variant 7 months following immunization. These results, together with our recent results from an identical study in non-human primates (NHPs), provide a comparative benchmarking of five clinically relevant vaccine adjuvants for their capacity to stimulate immunity to a subunit vaccine, demonstrating the capacity of adjuvanted SARS-CoV-2 subunit vaccines to provide durable protection against the B.1.351 variant. Furthermore, these results reveal differences between the widely-used C57BL/6 mouse strain and NHP animal models, highlighting the importance of species selection for future vaccine and adjuvant studies.

BackgroundVaccine efficacy against the B.1.351 variant following mRNA-1273 vaccination in humans has not been determined. Nonhuman primates (NHP) are a useful model for demonstrating whether mRNA-1273 mediates protection against B.1.351.MethodsNonhuman primates received 30 or 100 µg of mRNA-1273 as a prime-boost vaccine at 0 and 4 weeks, a single immunization of 30 µg at week 0, or no vaccine. Antibody and T cell responses were assessed in blood, bronchioalveolar lavages (BAL), and nasal washes. Viral replication in BAL and nasal swabs were determined by qRT-PCR for sgRNA, and histopathology and viral antigen quantification were performed on lung tissue post-challenge.ResultsEight weeks post-boost, 100 µg x2 of mRNA-1273 induced reciprocal ID50 neutralizing geometric mean titers against live SARS-CoV-2 D614G and B.1.351 of 3300 and 240, respectively, and 430 and 84 for the 30 µg x2 group. There were no detectable neutralizing antibodies against B.1351 after the single immunization of 30 µg. On day 2 following B.1.351 challenge, sgRNA in BAL was undetectable in 6 of 8 NHP that received 100 µg x2 of mRNA-1273, and there was a ∼2-log reduction in sgRNA in NHP that received two doses of 30 µg compared to controls. In nasal swabs, there was a 1-log10 reduction observed in the 100 µg x2 group. There was limited inflammation or viral antigen in lungs of vaccinated NHP post-challenge.ConclusionsImmunization with two doses of mRNA-1273 achieves effective immunity that rapidly controls lower and upper airway viral replication against the B.1.351 variant in NHP.

SARS-CoV-2 has infected over 160 million and caused more than 3 million deaths to date. Most individuals (>80%) have mild symptoms and recover in the outpatient setting, but detailed studies of immune responses have focused primarily on moderate to severe COVID-19. We deeply profiled the longitudinal immune response in individuals with mild COVID beginning with early time points post-infection (1-15 days) and proceeding through convalescence to >100 days after symptom onset. We correlated data from single cell analyses of peripheral blood cells, serum proteomics, virus-specific cellular and humoral immune responses, and clinical metadata. Acute infection was characterized by vigorous coordinated innate and adaptive activation, including an early cellular and proteomic signature that correlated with the amplitude of virus-specific humoral responses after day 30. We characterized signals associated with recovery and convalescence to define a new signature of inflammatory cytokines, gene expression, and chromatin accessibility that persists in individuals with post-acute sequelae of SARS-CoV-2 infection (PASC).

SARS-CoV-2 mutations may diminish vaccine-induced protective immune responses, and the durability of such responses has not been previously reported. Here, we present a comprehensive assessment of the impact of variants B.1.1.7, B.1.351, P.1, B.1.429, and B.1.526 on binding, neutralizing, and ACE2-blocking antibodies elicited by the vaccine mRNA-1273 over seven months. Cross-reactive neutralizing responses were rare after a single dose of mRNA-1273. At the peak of response to the second dose, all subjects had robust responses to all variants. Binding and functional antibodies against variants persisted in most subjects, albeit at low levels, for 6 months after the primary series of mRNA-1273. Across all assays, B.1.351 had the greatest impact on antibody recognition, and B.1.1.7 the least. These data complement ongoing studies of clinical protection to inform the potential need for additional boost vaccinations., One-Sentence Summary: Most mRNA-1273 vaccinated individuals maintained binding and functional antibodies against SARS-CoV-2 variants for 6 months.

SARS-CoV-2 has caused a devastating global pandemic. The recent emergence of SARS-CoV-2 variants that are less sensitive to neutralization by convalescent sera or vaccine-induced neutralizing antibody responses has raised concerns. A second wave of SARS-CoV-2 infections in India is leading to the expansion of SARS-CoV-2 variants. The B.1.617.1 variant has rapidly spread throughout India and to several countries throughout the world. In this study, using a live virus assay, we describe the neutralizing antibody response to the B.1.617.1 variant in serum from infected and vaccinated individuals. We found that the B.1.617.1 variant is 6.8-fold more resistant to neutralization by sera from COVID-19 convalescent and Moderna and Pfizer vaccinated individuals. Despite this, a majority of the sera from convalescent individuals and all sera from vaccinated individuals were still able to neutralize the B.1.617.1 variant. This suggests that protective immunity by the mRNA vaccines tested here are likely retained against the B.1.617.1 variant. As the B.1.617.1 variant continues to evolve, it will be important to monitor how additional mutations within the spike impact antibody resistance, viral transmission and vaccine efficacy.

BackgroundThe effects of pre-existing endemic human coronavirus (HCoV) immunity on SARS-CoV-2 serologic and clinical responses are incompletely understood.ObjectivesWe sought to determine the effects of prior exposure to HCoV Betacoronavirus HKU1 spike protein on serologic responses to SARS-CoV-2 spike protein after intramuscular administration in mice. We also sought to understand the baseline seroprevalence of HKU1 spike antibodies in healthy children and to measure their correlation with SARS-CoV-2 binding and neutralizing antibodies in children hospitalized with acute coronavirus disease 2019 (COVID-19) or multisystem inflammatory syndrome (MIS-C).MethodsGroups of 5 mice were injected intramuscularly with two doses of alum-adjuvanted HKU1 spike followed by SARS-CoV-2 spike; or the reciprocal regimen of SARS-Cov-2 spike followed by HKU1 spike. Sera collected 21 days following each injection was analyzed for IgG antibodies to HKU1 spike, SARS-CoV-2 spike, and SARS-CoV-2 neutralization. Sera from children hospitalized with acute COVID-19, MIS-C or healthy controls (n=14 per group) were analyzed for these same antibodies.ResultsMice primed with SARS-CoV-2 spike and boosted with HKU1 spike developed high titers of SARS-CoV-2 binding and neutralizing antibodies; however, mice primed with HKU1 spike and boosted with SARS-CoV-2 spike were unable to mount neutralizing antibodies to SARS-CoV-2. HKU1 spike antibodies were detected in all children with acute COVID-19, MIS-C, and healthy controls. Although children with MIS-C had significantly higher HKU1 spike titers than healthy children (GMT 37239 vs. 7551, P=0.012), these titers correlated positively with both SARS-CoV-2 binding (r=0.7577, PP=0.001) antibodies.ConclusionsPrior murine exposure to HKU1 spike protein completely impeded the development of neutralizing antibodies to SARS-CoV-2, consistent with original antigenic sin. In contrast, the presence of HKU1 spike IgG antibodies in children with acute COVID-19 or MIS-C was not associated with diminished neutralizing antibody responses to SARS-CoV-2.

Immune correlates of protection can be used as surrogate endpoints for vaccine efficacy. The nonhuman primate (NHP) model of SARS-CoV-2 infection replicates key features of human infection and may be used to define immune correlates of protection following vaccination. Here, NHP received either no vaccine or doses ranging from 0.3 – 100 μg of mRNA-1273, a mRNA vaccine encoding the prefusion-stabilized SARS-CoV-2 spike (S-2P) protein encapsulated in a lipid nanoparticle. mRNA-1273 vaccination elicited robust circulating and mucosal antibody responses in a dose-dependent manner. Viral replication was significantly reduced in bronchoalveolar lavages and nasal swabs following SARS-CoV-2 challenge in vaccinated animals and was most strongly correlated with levels of anti-S antibody binding and neutralizing activity. Consistent with antibodies being a correlate of protection, passive transfer of vaccine-induced IgG to naïve hamsters was sufficient to mediate protection. Taken together, these data show that mRNA-1273 vaccine-induced humoral immune responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.One-Sentence SummarymRNA-1273 vaccine-induced antibody responses are a mechanistic correlate of protection against SARS-CoV-2 infection in NHP.

Ending the COVID-19 pandemic will require long-lived immunity to SARS-CoV-2. Here, we evaluate 254 COVID-19 patients longitudinally up to 8 months and find durable broad-based immune responses. SARS-CoV-2 spike binding and neutralizing antibodies exhibit a bi-phasic decay with an extended half-life of >200 days suggesting the generation of longer-lived plasma cells. SARS-CoV-2 infection also boosts antibody titers to SARS-CoV-1 and common betacoronaviruses. In addition, spike-specific IgG+ memory B cells persist, which bodes well for a rapid antibody response upon virus re-exposure or vaccination. Virus-specific CD4+ and CD8+ T cells are polyfunctional and maintained with an estimated half-life of 200 days. Interestingly, CD4+ T cell responses equally target several SARS-CoV-2 proteins, whereas the CD8+ T cell responses preferentially target the nucleoprotein, highlighting the potential importance of including the nucleoprotein in future vaccines. Taken together, these results suggest that broad and effective immunity may persist long-term in recovered COVID-19 patients., Graphical abstract, Cohen et al. evaluate immune responses longitudinally in 254 COVID-19 patients over 8 months. SARS-CoV-2-specific binding and neutralizing antibodies exhibit biphasic decay, suggesting long-lived plasma cell generation. Memory B cells remain stable; CD4 and CD8 memory T cells are polyfunctional. Thus, broad and effective immunity may persist long-term following COVID-19.

The Omicron variant of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) exhibits reduced susceptibility to vaccine-induced neutralizing antibodies, requiring a boost to generate protective immunity. We assess the magnitude and short-term durability of neutralizing antibodies after homologous and heterologous boosting with mRNA and Ad26.COV2.S vaccines. All prime-boost combinations substantially increase the neutralization titers to Omicron, although the boosted titers decline rapidly within 2 months from the peak response compared with boosted titers against the prototypic D614G variant. Boosted Omicron neutralization titers are substantially higher for homologous mRNA vaccine boosting, and for heterologous mRNA and Ad26.COV2.S vaccine boosting, compared with homologous Ad26.COV2.S boosting. Homologous mRNA vaccine boosting generates nearly equivalent neutralizing activity against Omicron sublineages BA.1, BA.2, and BA.3 but modestly reduced neutralizing activity against BA.2.12.1 and BA.4/BA.5 compared with BA.1. These results have implications for boosting requirements to protect against Omicron and future variants of SARS-CoV-2. This trial was conducted under ClincalTrials.gov: NCT04889209.

The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies. We compared antibody binding and live virus neutralization of sera from naturally infected and Moderna-vaccinated individuals against two SARS-CoV-2 variants: B.1 containing the spike mutation D614G and the emerging B.1.351 variant containing additional spike mutations and deletions. Sera from acutely infected and convalescent COVID-19 patients exhibited a 3-fold reduction in binding antibody titers to the B.1.351 variant receptor-binding domain of the spike protein and a 3.5-fold reduction in neutralizing antibody titers against SARS-CoV-2 B.1.351 variant compared to the B.1 variant. Similar results were seen with sera from Moderna-vaccinated individuals. Despite reduced antibody titers against the B.1.351 variant, sera from infected and vaccinated individuals containing polyclonal antibodies to the spike protein could still neutralize SARS-CoV-2 B.1.351, suggesting that protective humoral immunity may be retained against this variant., Graphical abstract, In this study, Edara et al. (2021) report that, despite reduced antibody binding to the B.1.351 RBD, sera from infected (acute and convalescent) and Moderna (mRNA-1273)-vaccinated individuals were still able to neutralize the SARS-CoV-2 B.1.351 variant, suggesting that protective humoral immunity may be retained against this variant.

A combination of vaccination approaches will likely be necessary to fully control the SARS-CoV-2 pandemic. Here, we show that modified vaccinia Ankara (MVA) vectors expressing membrane anchored pre-fusion stabilized spike (MVA/S), but not secreted S1, induced strong neutralizing antibody responses against SARS-CoV-2 in mice. In macaques, the MVA/S vaccination induced strong neutralizing antibodies and CD8+ T cell responses, and showed protection from SARS-CoV-2 infection and virus replication in the lung as early as day 2 following intranasal or intratracheal challenge. Single-cell RNA sequencing analysis of lung cells at day 4 post-infection revealed that MVA/S vaccination also protected macaques from infection-induced inflammation and B cell abnormalities, and lowered induction of interferon stimulated genes. These results demonstrate that MVA/S vaccination induces both neutralizing antibodies and CD8+ T cells in the blood and lung and serves as a potential vaccine candidate against SARS-CoV-2., Graphical Abstract, Modified vaccinia Ankara (MVA) vector-based vaccines are attractive for their excellent safety and ability to induce long-lived humoral and cellular immunity in humans. Routhu et al. show that an MVA-based COVID-19 vaccine encoding prefusion-stabilized spike MVA/S induces strong neutralizing antibody and CD8+ T cell responses and protects macaques from SARS-CoV2 infection, immunopathology and infection-induced B cell abnormalities in the lung.

Adjuvanted soluble protein vaccines have been used extensively in humans for protection against various viral infections based on their robust induction of antibody responses. Here, soluble prefusion-stabilized spike trimers (preS dTM) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) were formulated with the adjuvant AS03 and administered twice to nonhuman primates (NHP). Binding and functional neutralization assays and systems serology revealed that NHP developed AS03-dependent multi-functional humoral responses that targeted multiple spike domains and bound to a variety of antibody FCreceptors mediating effector functionsin vitro. Pseudovirus and live virus neutralizing IC50titers were on average greater than 1000 and significantly higher than a panel of human convalescent sera. NHP were challenged intranasally and intratracheally with a high dose (3×106PFU) of SARS-CoV-2 (USA-WA1/2020 isolate). Two days post-challenge, vaccinated NHP showed rapid control of viral replication in both the upper and lower airways. Notably, vaccinated NHP also had increased spike-specific IgG antibody responses in the lung as early as 2 days post challenge. Moreover, vaccine-induced IgG mediated protection from SARS-CoV-2 challenge following passive transfer to hamsters. These data show that antibodies induced by the AS03-adjuvanted preS dTM vaccine are sufficient to mediate protection against SARS-CoV-2 and support the evaluation of this vaccine in human clinical trials.

In a phase 1 randomized, single-center clinical trial, inactivated influenza virus vaccine delivered through dissolvable microneedle patches (MNPs) was found to be safe and immunogenic. Here, we compare the humoral and cellular immunologic responses in a subset of participants receiving influenza vaccination by MNP to the intramuscular (IM) route of administration. We collected serum, plasma, and peripheral blood mononuclear cells in 22 participants up to 180 days post-vaccination. Hemagglutination inhibition (HAI) titers and antibody avidity were similar after MNP and IM vaccination, even though MNP vaccination used a lower antigen dose. MNPs generated higher neuraminidase inhibition (NAI) titers for all three influenza virus vaccine strains tested and triggered a larger percentage of circulating T follicular helper cells (CD4 + CXCR5 + CXCR3 + ICOS + PD-1+) compared to the IM route. Our study indicates that inactivated influenza virus vaccination by MNP produces humoral and cellular immune response that are similar or greater than IM vaccination.

The emergence of SARS-CoV-2 variants with mutations in the spike protein is raising concerns about the efficacy of infection- or vaccine-induced antibodies. We compared antibody binding and live virus neutralization of sera from naturally infected and Moderna-vaccinated individuals against two SARS-CoV-2 variants: B.1 containing the spike mutation D614G and the emerging B.1.351 variant containing additional spike mutations and deletions. Sera from acutely infected and convalescent COVID-19 patients exhibited a 3-fold reduction in binding antibody titers to the B.1.351 variant receptor-binding domain of the spike protein and a 3.5-fold reduction in neutralizing antibody titers against SARS-CoV-2 B.1.351 variant compared to the B.1 variant. Similar results were seen with sera from Moderna-vaccinated individuals. Despite reduced antibody titers against the B.1.351 variant, sera from infected and vaccinated individuals containing polyclonal antibodies to the spike protein could still neutralize SARS-CoV-2 B.1.351, suggesting that protective humoral immunity may be retained against this variant.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

The development of a portfolio of SARS-CoV-2 vaccines to vaccinate the global population remains an urgent public health imperative. Here, we demonstrate the capacity of a subunit vaccine under clinical development, comprising the SARS-CoV-2 Spike protein receptor-binding domain displayed on a two-component protein nanoparticle (RBD-NP), to stimulate robust and durable neutralizing antibody (nAb) responses and protection against SARS-CoV-2 in non-human primates. We evaluated five different adjuvants combined with RBD-NP including Essai O/W 1849101, a squalene-in-water emulsion; AS03, an alpha-tocopherol-containing squalene-based oil-in-water emulsion used in pandemic influenza vaccines; AS37, a TLR-7 agonist adsorbed to Alum; CpG 1018-Alum (CpG-Alum), a TLR-9 agonist formulated in Alum; or Alum, the most widely used adjuvant. All five adjuvants induced substantial nAb and CD4 T cell responses after two consecutive immunizations. Durable nAb responses were evaluated for RBD-NP/AS03 immunization and the live-virus nAb response was durably maintained up to 154 days post-vaccination. AS03, CpG-Alum, AS37 and Alum groups conferred significant protection against SARS-CoV-2 infection in the pharynges, nares and in the bronchoalveolar lavage. The nAb titers were highly correlated with protection against infection. Furthermore, RBD-NP when used in conjunction with AS03 was as potent as the prefusion stabilized Spike immunogen, HexaPro. Taken together, these data highlight the efficacy of the RBD-NP formulated with clinically relevant adjuvants in promoting robust immunity against SARS-CoV-2 in non-human primates.

Antibody responses against the SARS-CoV-2 Spike protein correlate with protection against COVID-19. Serum neutralizing antibodies appear early after symptom onset following SARS-CoV-2 infection and can last for several months. Similarly, the messenger RNA vaccine, mRNA-1273, generates serum neutralizing antibodies that are detected through at least day 119. However, the recent emergence of the B.1.1.7 variant has raised significant concerns about the breadth of these neutralizing antibody responses. In this study, we used a live virus neutralization assay to compare the neutralization potency of sera from infected and vaccinated individuals against a panel of SARS-CoV-2 variants, including SARS-CoV-2 B.1.1.7. We found that both infection- and vaccine-induced antibodies were effective at neutralizing the SARS-CoV-2 B.1.1.7 variant. These findings support the notion that in the context of the UK variant, vaccine-induced immunity can provide protection against COVID-19. As additional SARS-CoV-2 viral variants continue to emerge, it is crucial to monitor their impact on neutralizing antibody responses following infection and vaccination.

(1) Background: The influenza virus continues to cause significant annual morbidity and mortality. The overall efficacy of seasonal influenza vaccination is suboptimal, which is partly due to host immune factors. The effects of imprinting and repeated seasonal influenza vaccination were investigated to assess for immune factors and mechanisms that impact influenza vaccine responses. (2) Methods: Twenty participants were enrolled into a prospective pilot study based on birth cohort and seasonal influenza immunization history. Immunologic parameters were assessed over a six-month period after the seasonal influenza vaccine was administered. (3) Results: There was no significant imprinting effect, as measured by hemagglutination inhibition (HAI) fold change, HAI geometric mean titer (GMT) for Day 29 or Day 180 post-vaccination and antigen- specific antibody-secreting cells (ASC) for Day 8 post-vaccination. Individuals who had minimal prior seasonal influenza vaccination had a higher magnitude ASC response and a higher HAI fold change post-vaccination than individuals who were repeatedly vaccinated. (4) Conclusions: Repeated seasonal influenza vaccination resulted in a decreased fold change of the immune response, although individuals in this cohort tended to have high HAI titers at baseline that persisted after vaccination. Imprinting effects were not observed in this cohort. These host immune factors should be considered in the development of universal influenza vaccines. ClinicalTrials.gov Identifier: NCT03686514

The human immune response to eastern equine encephalitis virus (EEEV) infection is poorly characterized due to the rarity of infection. We examined the humoral and cellular immune response to EEEV acquired from an infected donor via liver transplantation. Both binding and highly neutralizing antibodies to EEEV as well as a robust EEEV-specific IgG memory B cell response were generated. Despite triple-drug immunosuppressive therapy, a virus-specific CD4+ T cell response, predominated by interferon-γ production, was generated. T cell epitopes on the E2 envelope protein were identified by interferon-γ ELISpot. Although these results are from a single person who acquired EEEV by a non-traditional mechanism, to our knowledge this work represents the first analysis of the human cellular immune response to EEEV.

There is a need for improved influenza vaccines. In this study we compared the antibody responses in humans after vaccination with an AS03-adjuvanted versus nonadjuvanted H5N1 avian influenza virus inactivated vaccine. Healthy young adults received two doses of either formulation 3 wk apart. We found that AS03 significantly enhanced H5 hemagglutinin (HA)-specific plasmablast and antibody responses compared to the nonadjuvanted vaccine. Plasmablast response after the first immunization was exclusively directed to the conserved HA stem region and came from memory B cells. Monoclonal antibodies (mAbs) derived from these plasmablasts had high levels of somatic hypermutation (SHM) and recognized the HA stem region of multiple influenza virus subtypes. Second immunization induced a plasmablast response to the highly variable HA head region. mAbs derived from these plasmablasts exhibited minimal SHM (naive B cell origin) and largely recognized the HA head region of the immunizing H5N1 strain. Interestingly, the antibody response to H5 HA stem region was much lower after the second immunization, and this suppression was most likely due to blocking of these epitopes by stem-specific antibodies induced by the first immunization. Taken together, these findings show that an adjuvanted influenza vaccine can substantially increase antibody responses in humans by effectively recruiting preexisting memory B cells as well as naive B cells into the response. In addition, we show that high levels of preexisting antibody can have a negative effect on boosting. These findings have implications toward the development of a universal influenza vaccine.

Zika virus (ZIKV) has become a global public health issue due to its teratogenicity and ability to cause Guillain-Barre syndrome in adults. Although anti-ZIKV envelope protein neutralizing antibodies correlate with protection, the non-neutralizing function of ZIKV antibodies including antibody-dependent cell-mediated cytotoxicity (ADCC) is incompletely understood. To study the role of ADCC antibodies during ZIKV infections, we generated a stably transfected, dual-reporter target cell line with inducible expression of a chimeric ZIKV prM-E protein on the cell surface as the target cell for the assay. By using this assay, nine of ten serum samples from ZIKV-infected patients had >20% ADCC killing of target cells, whereas none of the 12 healthy control sera had >10% ADCC killing. We also observed a time-dependent ADCC response in 2 patients with Zika. This demonstrates that this assay can detect ZIKV ADCC with high sensitivity and specificity, which could be useful for measurement of ADCC responses to ZIKV infection or vaccination.

The cellular immune responses elicited by an investigational vaccine against an emergent variant of influenza (H3N2v) are not fully understood. Twenty-five subjects, enrolled in an investigational influenza A/H3N2v vaccine study, who received two doses of vaccine 21 days apart, were included in a sub-study of cellular immune responses. H3N2v-specific plasmablasts were determined by ELISpot 8 days after each vaccine dose and H3N2v specific CD4+ T cells were quantified by intracellular cytokine and CD154 (CD40 ligand) staining before vaccination, 8 and 21 days after each vaccine dose. Results: 95% (19/20) and 96% (24/25) subjects had pre-existing H3N2v specific memory B, and T cell responses, respectively. Plasmablast responses at Day 8 after the first vaccine administration were detected against contemporary H3N2 strains and correlated with hemagglutination inhibition HAI (IgG: p = 0.018, IgA: p &lt, 0.001) and Neut (IgG: p = 0.038, IgA: p = 0.021) titers and with memory B cell frequency at baseline (IgA: r = 0.76, p &lt, 0.001, IgG: r = 0.74, p = 0.0001). The CD4+ T cells at Days 8 and 21 expanded after prime vaccination and this expansion correlated strongly with early post-vaccination HAI and Neut titers (p &le, 0.002). In an adult population, the rapid serological response observed after initial H3N2v vaccination correlates with post-vaccination plasmablasts and CD4+ T cell responses.

Background: Vaccinia is known to induce antibody and cellular responses. Plasmablast, circulating follicular helper T (cTFH) cells, cytokine-expressing CD4 T cells, and memory B cells were compared between subcutaneous (SC) and needle-free jet injection (JI) recipients of non-replicating modified vaccinia Ankara (MVA) vaccine. Methods: Vaccinia-na&iuml, ve adults received MVA SC or by JI on Days 1 and 29. Vaccinia-specific antibodies were quantified by plaque reduction neutralization test (PRNT) and enzyme-linked immunosorbent assay. Plasmablast, cTFH, and cytokine-expressing CD4 T cells were assessed on Days 1, 8, 15, 29, 36, 43 (cTFH and CD4+ only) and 57. Memory B cells were measured on Days 1 and 57. Results: Of the 36 enrolled subjects, only 22 received both vaccinations and had evaluable specimens after the second vaccine. Plasmablasts peaked one week after each vaccine. Day 15 plasmablasts correlated with peak PRNT titers. cTFH peaked on Days 8 and 36 and correlated with Day 36 plasmablasts. CD4+ peaked at Day 29 and one-third produced &ge, 2 cytokines. Day 57 memory B cells ranged from 0.1% to 0.17% of IgG-secreting B cells. Conclusions: This study provides insights into the cellular responses to non-replicating MVA, currently used as a vector for a variety of novel vaccines.

The COVID-19 pandemic has caused a global public health crisis of unprecedented proportions. SARS-CoV-2, the etiological agent of COVID-19, primarily infects the respiratory tract, leading to a range of symptoms including dry cough, fever, and shortness of breath. While many cases remain mild or asymptomatic, a subset progress to acute respiratory distress syndrome (ARDS) requiring ICU admission and ventilation support. Rapid progress has been made to understand the pathogenesis and immune response to SARS-CoV-2 infection; however, there remain a number of unanswered questions. Recent evidence has indicated that mild cases of COVID-19 are controlled by an effective innate immune response. However, in moderate and severe cases, the immune response to infection appears to be dysfunctional leading to low levels of interferon and high inflammatory responses. Here, we examined a cohort of patients admitted to the Emory University Hospital with severe COVID-19. Patients had variable neutralization titers measured by FRNT assay, which did not correlate age or disease severity. Flow cytometry immune subset analysis, cytokine and chemokine quantification, and single cell RNA sequencing (scRNAseq) revealed an inflammatory phenotype including a significant shift in the myeloid compartment compared to healthy donors. Numbers of pDCs and non-classical (CD14−CD16+) monocytes in the blood were decreased, while intermediate monocytes (CD14+CD16+) cells were increased. Additionally, stimulated CD14+ cells from healthy donors were able to limit SARS-CoV-2 infection in a human airway epithelial culture system. This suggests a role for monocytes to combat infection by SARS-CoV-2 that may be dysregulated in severe patients.

From September 2014 to April 2015, 6 persons who had occupational exposures to Zaire ebolavirus in West Africa received investigational agent rVSV-ZEBOV or TKM-100802 for postexposure prophylaxis and were monitored in the United States. All patients experienced self-limited symptoms after postexposure prophylaxis; none developed Ebola virus disease.

The re-emergence of Zika virus (ZIKV) in the western hemisphere has most significantly affected dengue virus (DENV) endemic regions. Due to the geographical overlap between these two closely related flaviviruses, numerous individuals who suffered ZIKV infection during recent outbreaks may have also previously been exposed to DENV. As such, the impact of pre-existing dengue immunity on immune responses to ZIKV has been an area of focused research and interest. To understand how B cell responses to a ZIKV infection may be modulated by prior dengue exposures, we compared and contrasted plasmablast repertoire and specificity between two ZIKV-infected individuals, one dengue-na&iuml, ve (ZK018) and the other dengue-experienced (ZK016). In addition to examining serological responses, we generated 59 patient plasmablast-derived monoclonal antibodies (mAbs) to define the heterogeneity of the early B cell response to ZIKV. Both donors experienced robust ZIKV-induced plasmablast expansions early after infection, with comparable mutational frequencies in their antibody variable genes. However, notable differences were observed in plasmablast clonality and functional reactivity. Plasmablasts from the dengue-experienced donor ZK016 included cells with shared clonal origin, while ZK018 mAbs were entirely clonally unrelated. Both at the mAb and plasma level, ZK016 antibodies displayed extensive cross-reactivity to DENV1-4, and preferentially neutralized DENV compared to ZIKV. In contrast, the neutralization activity of ZK018 mAbs was primarily directed towards ZIKV, and fewer mAbs from this donor were cross-reactive, with the cross-reactive phenotype largely limited to fusion loop-specific mAbs. ZK016 antibodies caused greater enhancement of DENV2 infection of FcR&gamma, expressing cells overall compared to ZK018, with a striking difference at the plasma level. Taken together, these data strongly suggest that the breadth and protective capacity of the initial antibody responses after ZIKV infection may depend on the dengue immune status of the individual. These findings have implications for vaccine design, given the likelihood that future epidemics will involve both dengue-experienced and na&iuml, ve populations.

Background Clinical, virologic, and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US subjects with suspected ZIKV infection were enrolled. Clinical data and specimens were prospectively collected for ZIKV RNA detection and serologic and cellular assays. Confirmed ZIKV infection (cases) and ZIKV-negative (controls) subjects were compared. Dengue-experienced and dengue-naïve cases were also compared. Results We enrolled 45 cases and 14 controls. Commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%), and arthralgia (82.2% and 54.5%), respectively. The sensitivity (94%) and duration of infection detection (80% positivity at 65–79 days after disease onset) by polymerase chain reaction were highest in whole-blood specimens. ZIKV-neutralizing antibodies had a half-life of 105 days and were significantly higher in dengue virus–experienced cases than naïve ones (P = .046). In intracellular cytokine staining assays, the ZIKV proteins targeted most often by peripheral blood mononuclear cells from cases were structural proteins C and E for CD4+ T cells and nonstructural proteins NS3, NS5, and NS4B for CD8+ T cells. Conclusions ZIKV RNA detection was more frequent and prolonged in whole-blood specimens. Immunoglobulin G (IgG) and neutralizing antibodies, but not IgM, were influenced by prior dengue infection. Robust cellular responses to E and nonstructural proteins have potential vaccine development implications.

Understanding the immune response during acute Zika in humans will aid vaccine design and testing. In 5 acute patients, including 2 pregnant women, viral levels and innate, T-, and B-cell responses against Zika or dengue viruses are described., Background There is an urgent need for studies of viral persistence and immunity during human Zika infections to inform planning and conduct of vaccine clinical trials. Methods In 5 returned US travelers with acute symptomatic Zika infection, clinical features, viral RNA levels, and immune responses were characterized. Results Two pregnant, flavivirus-experienced patients had viral RNA persist in plasma for >44 and >26 days. Three days after symptom onset, transient increases in proinflammatory monocytes began followed at 5 days by transient decreases in myeloid dendritic cells. Anti-Zika virus immunoglobulin M was detected at day 7 after symptom onset, persisted beyond 103 days, and remained equivocal through day 172. Zika virus–specific plasmablasts and neutralizing antibodies developed quickly; dengue virus–specific plasmablasts and neutralizing antibodies at high titers developed only in flavivirus-experienced patients. Zika virus– and dengue virus–specific memory B cells developed in both flavivirus-naive and -experienced patients. CD4+ T cells were moderately activated and produced antiviral cytokines after stimulation with Zika virus C, prM, E, and NS5 peptides in 4/4 patients. In contrast, CD8+ T cells were massively activated, but virus-specific cells that produced cytokines were present in only 2/4 patients assessed. Conclusions Acute infections with Zika virus modulated antigen-presenting cell populations early. Flavivirus-experienced patients quickly recalled cross-reactive MBCs to secrete antibodies. Dengue virus–naive patients made little dengue-specific antibody but developed MBCs that cross-reacted against dengue virus. Zika virus–specific functional CD4+ T cells were readily detected, but few CD8+ T cells specific for the tested peptides were found.

Background The host immune response to influenza vaccination can be affected by prior imprinting to a specific influenza strain based on birth cohort and prior influenza vaccination history. Understanding the underlying immune mechanisms is essential to development of an improved seasonal vaccine and an effective universal influenza vaccine. Methods This is a prospective pilot study, with a total of 20 subjects in either the H3N2 cohort (N = 10, born 1968–1977) or the H1N1 cohort (N = 10, born 1948–1957). Each cohort was further stratified by subjects who have received the influenza vaccine < 2 or ≥ 3 of the past 5 years. The FDA-approved quadrivalent 2018–19 influenza vaccine (containing A(H1N1), an A/Michigan/45/2015-like virus; A(H3N2), an A/Singapore/INFIMH-16–0019/2016-like virus; B/Colorado/06/2017-like virus; and B/Phuket/3073/2013-like virus) was administered on Day 1. Demographic information included age, gender, ethnicity, and BMI. HAI titers for each component of the vaccine were obtained at baseline, 29 days post-vaccination, and 180 days post-vaccination. HAI fold-change and HAI geometric mean titers (GMT) were analyzed. Results There was no significant difference between H1N1 or H3N2 HAI fold-change in the H3N2 birth cohort (P = 0.7496) or in the H1N1 birth cohort (P = 0.8237), Figure A. Comparing HAI fold-change for the repeatedly vs. minimally vaccinated groups, there was a significant higher fold change in the minimally vaccinated group (H1N1 HAI (P = 0.002) and H3N2 HAI (P < 0.0001), Figure B). GMT was higher at baseline for the repeatedly vaccinated group (H1N1, 70; H3N2, 98; vs. H1N1, 30; H3N2, 21 for the minimally vaccinated group); however, the GMT for the minimally vaccinated group was higher at day 29 (H1N1, 172; H3N2, 184; vs. H1N1, 422; H3N2, 299 for the minimally vaccinated group; Figure C). HAI titers and analysis at day 180 post vaccination are in progress. Conclusion There was no evidence of an imprinting effect by birth cohort for HAI titer magnitudes, even when stratified by vaccination history. There was a significantly higher HAI fold change for individuals who had received minimal influenza vaccinations in the past 5 years at 29 days post-vaccination. Individuals who had repeated vaccinations in the last 5 years had higher HAI GMT at baseline. Disclosures Nadine Rouphael, MD, Merck: I conduct as Emory PI the PNEUMO MERCK study at Emory, Research Grant; Pfizer: I conduct as co-PI the RSV PFIZER study at Emory, Research Grant; Sanofi-Pasteur: I conducted as Emory PI the CDIFFENSE trial at Emory, Research Grant.

Despite the widespread outbreak, few cases of Zika virus associated with cardiac manifestations have been described. We present a case of pericarditis in the setting of an acute, symptomatic Zika virus infection in a traveler returning from St. Thomas. Clinicians should be alert for this potential complication of Zika virus infection.

During the current Zika virus (ZIKV) outbreak, acute symptomatic ZIKV infection in adults appears to be a mild-to-moderate, self-limited illness. We present a case of ZIKV rash illness that improved and then relapsed without repeat exposure to ZIKV. Clinicians should be alert for relapses in patients with ZIKV infection.

Infection with Zika virus is an emerging public health crisis. We observed prolonged detection of virus RNA in vaginal mucosal swab specimens and whole blood for a US traveler with acute Zika virus infection who had visited Honduras. These findings advance understanding of Zika virus infection and provide data for additional testing strategies.

Summary Background Microneedle patches provide an alternative to conventional needle-and-syringe immunisation, and potentially offer improved immunogenicity, simplicity, cost-effectiveness, acceptability, and safety. We describe safety, immunogenicity, and acceptability of the first-in-man study on single, dissolvable microneedle patch vaccination against influenza. Methods The TIV-MNP 2015 study was a randomised, partly blinded, placebo-controlled, phase 1, clinical trial at Emory University that enrolled non-pregnant, immunocompetent adults from Atlanta, GA, USA, who were aged 18–49 years, naive to the 2014–15 influenza vaccine, and did not have any significant dermatological disorders. Participants were randomly assigned (1:1:1:1) to four groups and received a single dose of inactivated influenza vaccine (fluvirin: 18 μg of haemagglutinin per H1N1 vaccine strain, 17 μg of haemagglutinin per H3N2 vaccine strain, and 15 μg of haemagglutinin per B vaccine strain) (1) by microneedle patch or (2) by intramuscular injection, or received (3) placebo by microneedle patch, all administered by an unmasked health-care worker; or received a single dose of (4) inactivated influenza vaccine by microneedle patch self-administered by study participants. A research pharmacist prepared the randomisation code using a computer-generated randomisation schedule with a block size of 4. Because of the nature of the study, participants were not masked to the type of vaccination method (ie, microneedle patch vs intramuscular injection). Primary safety outcome measures are the incidence of study product-related serious adverse events within 180 days, grade 3 solicited or unsolicited adverse events within 28 days, and solicited injection site and systemic reactogenicity on the day of study product administration through 7 days after administration, and secondary safety outcomes are new-onset chronic illnesses within 180 days and unsolicited adverse events within 28 days, all analysed by intention to treat. Secondary immunogenicity outcomes are antibody titres at day 28 and percentages of seroconversion and seroprotection, all determined by haemagglutination inhibition antibody assay. The trial is completed and registered with ClinicalTrials.gov, number NCT02438423. Findings Between June 23, 2015, and Sept 25, 2015, 100 participants were enrolled and randomly assigned to a group. There were no treatment-related serious adverse events, no treatment-related unsolicited grade 3 or higher adverse events, and no new-onset chronic illnesses. Among vaccinated groups (vaccine via health-care worker administered microneedle patch or intramuscular injection, or self-administered microneedle patch), overall incidence of solicited adverse events (n=89 vs n=73 vs n=73) and unsolicited adverse events (n=18 vs n=12 vs n=14) were similar. Reactogenicity was mild, transient, and most commonly reported as tenderness (15 [60%] of 25 participants [95% CI 39–79]) and pain (11 [44%] of 25 [24–65]) after intramuscular injection; and as tenderness (33 [66%] of 50 [51–79]), erythema (20 [40%] of 50 [26–55]), and pruritus (41 [82%] of 50 [69–91]) after vaccination by microneedle patch application. The geometric mean titres were similar at day 28 between the microneedle patch administered by a health-care worker versus the intramuscular route for the H1N1 strain (1197 [95% CI 855–1675] vs 997 [703–1415]; p=0·5), the H3N2 strain (287 [192–430] vs 223 [160–312]; p=0·4), and the B strain (126 [86–184] vs 94 [73–122]; p=0·06). Similar geometric mean titres were reported in participants who self-administered the microneedle patch (all p>0·05). The seroconversion percentages were significantly higher at day 28 after microneedle patch vaccination compared with placebo (all p 0·01). Interpretation Use of dissolvable microneedle patches for influenza vaccination was well tolerated and generated robust antibody responses. Funding National Institutes of Health.

Since 2011, over 300 human cases of infection, especially in exposed children, with the influenza A H3N2 variant (H3N2v) virus that circulates in swine in the US have been reported. The structural and genetic basis for the lack of protection against H3N2v induced by vaccines containing seasonal H3N2 antigens is poorly understood. We isolated 17 human monoclonal antibodies (mAbs) that neutralized H3N2v virus from subjects experimentally immunized with an H3N2v candidate vaccine. Six mAbs exhibited very potent neutralizing activity (IC50 < 200 ng/ml) against the H3N2v virus but not against current human H3N2 circulating strains. Fine epitope mapping and structural characterization of antigen-antibody complexes revealed that H3N2v specificity was attributable to amino acid polymorphisms in the 150-loop and the 190-helix antigenic sites on the hemagglutinin protein. H3N2v-specific antibodies also neutralized human H3N2 influenza strains naturally circulating between 1995 and 2005. These results reveal a high level of antigenic relatedness between the swine H3N2v virus and previously circulating human strains, consistent with the fact that early human H3 seasonal strains entered the porcine population in the 1990s and reentered the human population, where they had not been circulating, as H3N2v about a decade later. The data also explain the increased susceptibility to H3N2v viruses in young children, who lack prior exposure to human seasonal strains from the 1990s.

Vaccine regimens using different agents for priming and boosting have become popular for enhancing T cell and Ab responses elicited by candidate HIV/AIDS vaccines. Here we use a simian model to evaluate immunogenicity and protective efficacy of a recombinant modified vaccinia Ankara (MVA) vaccine in the presence and absence of a recombinant DNA prime. The simian vaccines and regimens represent prototypes for candidate HIV vaccines currently undergoing clinical testing.Recombinant DNA and MVA immunogens expressed simian immunodeficiency virus (SIV)mac239 Gag, PR, RT, and Env sequences. Vaccine schedules tested inoculations of MVA at months 0, 2, and 6 (MMM regimen) or priming with DNA at months 0 and 2 and boosting with MVA at months 4 and 6 (DDMM regimen). Twelve weekly rectal challenges with the heterologous SIV smE660 were initiated at 6 months following the last immunization.Both regimens elicited similar 61-64% reductions in the per challenge risk of SIVsmE660 transmission despite raising different patterns of immune responses. The DDMM regimen elicited higher magnitudes of CD4 T cells whereas the MMM regimen elicited higher titers and greater avidity Env-specific IgG and more frequent and higher titer SIV-specific IgA in rectal secretions. Both regimens elicited similar magnitudes of CD8 T cells. Magnitudes of T cell responses, specific activities of rectal IgA Ab, and the tested specificities for neutralization and antibody-dependent cellular cytotoxicity did not correlate with risk of infection. However, the avidity of Env-specific IgG had a strong correlation with the per challenge risk of acquisition, but only for the DDMM group.We conclude that for the tested immunogens in rhesus macaques, the simpler MMM regimen is as protective as the more complex DDMM regimen.

A simian immunodeficiency virus (SIV) vaccine coexpressing granulocyte-macrophage colony stimulating factor (GM-CSF) prevented infection in 71% of macaques that received 12 rectal challenges. The SIVsmE660 challenge had the tropism of incident human immunodeficiency virus (HIV) infections and a similar genetic distance from the SIV239 vaccine as intraclade HIV isolates. The heterologous prime-boost vaccine regimen used recombinant DNA for priming and recombinant modified vaccinia Ankara for boosting. Co-expression of GM-CSF in the DNA prime enhanced the avidity of elicited immunoglobulin G for SIV envelope glycoproteins, the titers of neutralizing antibody for easy-to-neutralize SIV isolates, and antibody-dependent cellular cytotoxicity. Impressively, the co-expressed GM-CSF increased vaccine-induced prevention of infection from 25% in the non–GM-CSF co-expressing vaccine group to 71% in the GM-CSF co-expressing vaccine group. The prevention of infection showed a strong correlation with the avidity of the elicited Env-specific antibody for the Env of the SIVsmE660 challenge virus (r = 0.9; P < .0001).

Background. Recombinant DNA and modified vaccinia virus Ankara (rMVA) vaccines represent a promising approach to an HIV/AIDS vaccine. This Phase 1 clinical trial compared the safety and immunogenicity of a rMVA vaccine administered with and without DNA vaccine priming Methods. GeoVax pGA2/JS7 DNA (D) and MVA/HIV62 (M) vaccines encode noninfectious virus-like particles. Intramuscular needle injections were used to deliver placebo, 2 doses of DNA followed by 2 doses of rMVA (DDMM), one dose of DNA followed by 2 doses of rMVA (DMM), or 3 doses of rMVA (MMM) to HIV-seronegative participants. Results. Local and systemic symptoms were mild or moderate. Immune response rates for CD4 + and CD8 + T cells were highest in the DDMM group and lowest in the MMM group (77% vs 43% CD4 + and 42% vs 17% CD8 +). In contrast, response rates for Env binding and neutralizing Ab were highest in the MMM group. The DMM group had intermediate response rates. A 1/10th-dose DDMM regimen induced similar T cell but reduced Ab response rates compared with the full-dose DDMM. Conclusions. MVA62 was well tolerated and elicited different patterns of T cell and Ab responses when administered alone or in combination with the JS7 DNA vaccine.

Background The clinical, virologic and immunologic characteristics of Zika virus (ZIKV) infections in US patients are poorly defined. Methods US patients with suspected Zika virus (ZIKV) infection were enrolled and clinical data and specimens were prospectively collected. Body fluids were tested for ZIKV RNA by PCR and blood was tested using serologic and cellular immune assays. Findings from those with confirmed ZIKV infections (cases) and ZIKV-negative controls were compared. Results We enrolled 45 cases and 14 controls. The most commonly reported symptoms among cases and controls were maculopapular rash (97.8% and 81.8%), fatigue (86.7% and 81.8%) and arthralgia (82.2% and 54.5%), respectively. The sensitivity and duration of detection by PCR were highest in whole blood samples (94% of 35 cases who had samples collected up to day 79 post illness onset were positive); strikingly, 84% of those were still positive at 65–79 days post illness onset (Figure 1). ZIKV neutralizing antibodies were detected in all cases and none of the controls, and titers were significantly higher in dengue virus (DENV)-experienced subjects than in DENV-naïve ones (Figure 2). Among cases, anti-ZIKV IgG antibodies were also significantly higher in DENV-experienced patients, while anti-ZIKV IgM antibodies were no higher in DENV-experienced compared with naïve ones. Using intracellular cytokine staining, the highest frequencies of T cells producing IFN-γ, IL-2 and/or TNF-α were against the NS1, NS3, and NS5 proteins for CD4+ T cells, and against the E, NS3, and NS5 proteins for CD8+ T cells (Figure 3). Conclusion Detection of ZIKV RNA was more frequent and much more prolonged in whole blood samples compared with other body fluids. Diagnostic molecular assays on this easily obtained fluid should be prioritized for point-of-care development. Robust cellular responses to E, NS3 and NS5 proteins could have implications for vaccine development. Disclosures R. L. Atmar, Takeda Vaccines, Inc.: Investigator, Research grant.

A major challenge for human immunodeficiency virus (HIV)/AIDS vaccines is the elicitation of anti-Env antibodies (Ab) capable of neutralizing the diversity of isolates in the pandemic. Here, we show that high-avidity, but nonneutralizing, Abs can have an inverse correlation with peak postchallenge viremia for a heterologous challenge. Vaccine studies were conducted in rhesus macaques using DNA priming followed by modified vaccinia Ankara boosting with HIV type 1 (HIV-1) immunogens that express virus-like particles displaying CCR5-tropic clade B (strain ADA) or clade C (IN98012) Envs. Rhesus granulocyte-macrophage colony-stimulating factor was used as an adjuvant for enhancing the avidity of anti-Env Ab responses. Challenge was with simian/human immunodeficiency virus (SHIV)-162P3, a CCR5-tropic clade B chimera of SIV and HIV-1. Within the groups receiving the clade B vaccine, a strong inverse correlation was found between the avidity of anti-Env Abs and peak postchallenge viremia. This correlation required the use of native but not gp120 or gp140 forms of Env for avidity assays. The high-avidity Ab elicited by the ADA Env had excellent breadth for the Envs of incident clade B but not clade C isolates, whereas the high-avidity Ab elicited by the IN98012 Env had excellent breadth for incident clade C but not clade B isolates. High-avidity Ab elicited by a SHIV vaccine with a dual-tropic clade B Env (89.6) had limited breadth for incident isolates. Our results suggest that certain Envs can elicit nonneutralizing but high-avidity Ab with broad potential for blunting incident infections of the same clade.

Chronic immunodeficiency virus infections are characterized by dysfunctional cellular and humoral antiviral immune responses. As such, immune modulatory therapies that enhance and/or restore the function of virus-specific immunity may protect from disease progression. Here we investigate the safety and immune restoration potential of blockade of the co-inhibitory receptor programmed death 1 (PD-1) during chronic simian immunodeficiency virus (SIV) infection in macaques. We demonstrate that PD-1 blockade using an antibody to PD-1 is well tolerated and results in rapid expansion of virus-specific CD8 T cells with improved functional quality. This enhanced T-cell immunity was seen in the blood and also in the gut, a major reservoir of SIV infection. PD-1 blockade also resulted in proliferation of memory B cells and increases in SIV envelope-specific antibody. These improved immune responses were associated with significant reductions in plasma viral load and also prolonged the survival of SIV-infected macaques. Blockade was effective during the early (week 10) as well as late ( approximately week 90) phases of chronic infection even under conditions of severe lymphopenia. These results demonstrate enhancement of both cellular and humoral immune responses during a pathogenic immunodeficiency virus infection by blocking a single inhibitory pathway and identify a novel therapeutic approach for control of human immunodeficiency virus infections.

HIV incidence rates are higher among black men who have sex with men (BMSM) as compared with MSM of other race/ethnicities in the USA. We found that blood memory CD4 cells from BMSM express higher levels of α4β7, the gut-homing integrin, compared with white MSM. Higher expression of α4β7 on blood CD4 cells correlated with higher percentage of proliferating CD4α4β7 cells in rectal tissue suggesting increased trafficking of potential HIV target cells to rectal mucosa could increase HIV susceptibility among BMSM.

Single intradermal or intramuscular inoculations of GM-CSF DNA with the DNA prime for a simian–human immunodeficiency virus (SHIV)-89.6 vaccine, which consists of DNA priming followed by modified vaccinia Ankara (MVA) boosting, increased protection of both the blood and intestines against the acute phase of an intrarectal SHIV-89.6P challenge. GM-CSF appeared to contribute to protection by enhancing two antibody responses: the avidity maturation of anti-Env IgG in blood (p=

The clinical product for a clade B HIV DNA/MVA vaccine expressing Gag, Pol, and Env has been tested for immunogenicity in macaques. Responding T cells were at the threshold for detection following DNA priming at weeks 0 and 8 but underwent sharp expansions and contractions following MVA boosting at weeks 16 and 24. Both CD4 and CD8 T cell responses had high frequencies of cytokine coproducing cells with >50% of the memory cells coproducing multiple cytokines including IL-2. The highest responses were elicited to Gag, followed by Env and then Pol. In two of six macaques, the vaccine also elicited low levels of neutralizing Ab for easy to neutralize clade B isolates.

Primary keratinocytes immortalized by human papillomaviruses (HPVs), along with HPV-induced cervical carcinoma cell lines, are excellent models for investigating neoplastic progression to cancer. By simultaneously visualizing viral DNA and nascent viral transcripts in interphase nuclei, we demonstrated for the first time a selection for a single dominant papillomavirus transcription center or domain (PVTD) independent of integrated viral DNA copy numbers or loci. The PVTD did not associate with several known subnuclear addresses but was almost always perinucleolar. Silent copies of the viral genome were activated by growth in the DNA methylation inhibitor 5-azacytidine. HPV-immortalized keratinocytes supertransduced with HPV oncogenes and selected for marker gene coexpression underwent crisis, and the surviving cells transcribed only the newly introduced genes. Thus, transcriptional selection in response to environmental changes is a dynamic process to achieve optimal gene expression for cell survival. This phenomenon may be critical in clonal selection during carcinogenesis. Examination of HPV-associated cancers supports this hypothesis.