Protective Role of NS1-Specific Antibodies in the Immune Response to Dengue Virus through Antibody-Dependent Cellular Cytotoxicity.

BACKGROUND: Dengue virus (DENV) non-structural protein 1 (NS1) has multiple functions within infected cells, on the cell surface, and in secreted form, and is highly immunogenic. Immunity from previous DENV infections is known to exert both positive and negative effects on subsequent DENV infections, but the contribution of NS1-specific antibodies to these effects is incompletely understood. METHODS: We investigated the functions of NS1-specific antibodies and their significance in DENV infection. We analyzed plasma samples collected in a prospective cohort study prior to symptomatic or subclinical secondary DENV infection. We measured binding to purified recombinant NS1 protein and to NS1-expressing CEM cells, antibody-mediated NK cell activation by plate-bound NS1 protein, and antibody-dependent cellular cytotoxicity (ADCC) of NS1-expressing target cells. RESULTS: We found that antibody responses to NS1 were highly serotype-cross-reactive and that subjects who experienced subclinical DENV infection had significantly higher antibody responses to NS1 in pre-infection plasma than subjects who experienced symptomatic infection. We observed strong positive correlations between antibody binding and NK activation. CONCLUSIONS: These findings demonstrate the involvement of NS1-specific antibodies in ADCC and provide evidence for a protective effect of NS1-specific antibodies in secondary DENV infection.

Temporally integrated single cell RNA sequencing analysis of PBMC from experimental and natural primary human DENV-1 infections.

Dengue human infection studies present an opportunity to address many longstanding questions in the field of flavivirus biology. However, limited data are available on how the immunological and transcriptional response elicited by an attenuated challenge virus compares to that associated with a wild-type DENV infection. To determine the kinetic transcriptional signature associated with experimental primary DENV-1 infection and to assess how closely this profile correlates with the transcriptional signature accompanying natural primary DENV-1 infection, we utilized scRNAseq to analyze PBMC from individuals enrolled in a DENV-1 human challenge study and from individuals experiencing a natural primary DENV-1 infection. While both experimental and natural primary DENV-1 infection resulted in overlapping patterns of inflammatory gene upregulation, natural primary DENV-1 infection was accompanied with a more pronounced suppression in gene products associated with protein translation and mitochondrial function, principally in monocytes. This suggests that the immune response elicited by experimental and natural primary DENV infection are similar, but that natural primary DENV-1 infection has a more pronounced impact on basic cellular processes to induce a multi-layered anti-viral state.

Transmission of SARS-CoV-2 among recruits in a US Army training environment: a brief report.

BACKGROUND: In 2020, preventive measures were implemented to mitigate the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among 600-700 recruits arriving weekly at a basic combat training (BCT) facility in the southern United States. Trainees were sorted into companies and platoons (cocoons) at arrival, tested, quarantined for 14 days with daily temperature and respiratory-symptom monitoring and retested before release into larger groups for training where symptomatic testing was conducted. Nonpharmaceutical measures, such as masking, and social distancing, were maintained throughout quarantine and BCT. We assessed for SARS-CoV-2 transmission in the quarantine milieu. METHODS: Nasopharyngeal (NP) swabs were collected at arrival and at the end of quarantine and blood specimens at both timepoints and at the end of BCT. Epidemiological characteristics were analyzed for transmission clusters identified from whole-genome sequencing of NP samples. RESULTS: Among 1403 trainees enrolled from 25 August to 7 October 2020, epidemiological analysis identified three transmission clusters (n = 20 SARS-CoV-2 genomes) during quarantine, which spanned five different cocoons. However, SARS-CoV-2 incidence decreased from 2.7% during quarantine to 1.5% at the end of BCT; prevalence at arrival was 3.3%. CONCLUSIONS: These findings suggest layered SARS-CoV-2 mitigation measures implemented during quarantine minimized the risk of further transmission in BCT.

Genomic surveillance of SARS-CoV-2 in US military compounds in Afghanistan reveals multiple introductions and outbreaks of Alpha and Delta variants.

BACKGROUND: With the emergence and spread of SARS-CoV-2 variants, genomic epidemiology and surveillance have proven invaluable tools for variant tracking. Here, we analyzed SARS-CoV-2 samples collected from personnel located at the US/NATO bases across Afghanistan. RESULTS: Sequencing and phylogenetic analyses revealed at least 16 independent introductions of SARS-CoV-2 into four of these relatively isolated compounds during April and May 2021, including multiple introductions of Alpha and Delta variants. Four of the introductions resulted in sustained spread of the virus within, and in two cases between, the compounds. Three of these outbreaks, one Delta and two Alpha, occurred simultaneously. CONCLUSIONS: Even in rigorously controlled and segregated environments, SARS-CoV-2 introduction and spread may occur frequently.

Immunogenicity of a Live-Attenuated Dengue Vaccine Using a Heterologous Prime-Boost Strategy in a Phase 1 Randomized Clinical Trial.

BACKGROUND: Dengue is a global health problem and the development of a tetravalent dengue vaccine with durable protection is a high priority. A heterologous prime-boost strategy has the advantage of eliciting immune responses through different mechanisms and therefore may be superior to homologous prime-boost strategies for generating durable tetravalent immunity. METHODS: In this phase 1 first-in-human heterologous prime-boost study, 80 volunteers were assigned to 4 groups and received a tetravalent dengue virus (DENV-1-4) purified inactivated vaccine (TDENV-PIV) with alum adjuvant and a tetravalent dengue virus (DENV-1-4) live attenuated vaccine (TDENV-LAV) in different orders and dosing schedules (28 or 180 days apart). RESULTS: All vaccination regimens had acceptable safety profiles and there were no vaccine-related serious adverse events. TDEN-PIV followed by TDEN-LAV induced higher neutralizing antibody titers and a higher rate of tetravalent seroconversions compared to TDEN-LAV followed by TDEN-PIV. Both TDEN-PIV followed by TDEN-LAV groups demonstrated 100% tetravalent seroconversion 28 days following the booster dose, which was maintained for most of these subjects through the day 180 measurement. CONCLUSIONS: A heterologous prime-boost vaccination strategy for dengue merits additional evaluation for safety, immunogenicity, and potential for clinical benefit. CLINICAL TRIALS REGISTRATION: NCT02239614.

A Phase 1, Open-Label Assessment of a Dengue Virus-1 Live Virus Human Challenge Strain.

BACKGROUND: Dengue human infection models (DHIM) have been used as a safe means to test the viability of prophylaxis and therapeutics. METHODS: A phase 1 study of 12 healthy adult volunteers using a challenge virus, DENV-1-LVHC strain 45AZ5, was performed. A dose escalating design was used to determine the safety and performance profile of the challenge virus. Subjects were evaluated extensively until 28 days and then out to 6 months. RESULTS: Twelve subjects received the challenge virus: 6 with 0.5 mL of 6.5 × 103 plaque-forming units (PFU)/mL (low-dose group) and 6 with 0.5 mL of 6.5 × 104 PFU/mL (mid-dose group). All except 1 in the low-dose group developed detectable viremia. For all subjects the mean incubation period was 5.9 days (range 5-9 days) and mean time of viremia was 6.8 days (range 3-9 days). Mean peak for all subjects was 1.6 × 107 genome equivalents (GE)/mL (range 4.6 × 103 to 5 × 107 GE/mL). There were no serious adverse events or long-term safety signals noted. CONCLUSIONS: We conclude that DENV-1-LVHC was well-tolerated, resulted in an uncomplicated dengue illness, and may be a suitable DHIM for therapeutic and prophylactic product testing. CLINICAL TRIALS REGISTRATION: NCT02372175.

The Effects of Japanese Encephalitis Vaccine and Accelerated Dosing Scheduling on the Immunogenicity of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine: A Phase II, Randomized, Open-Label, Single-Center Trial in Adults Aged 18 to 45 Years in the United States.

BACKGROUND: Dengue is a global health problem requiring an effective, safe dengue vaccine. METHODS: We report the results of a phase II, randomized, open-label, single-center trial in adults aged 18 to 45 years in the United States designed to explore the effects of the Chimeric Yellow Fever Derived Tetravalent Dengue Vaccine (CYD-TDV, Dengvaxia) when administered on its designated schedule (months 0, 6, and 12) or on an accelerated dosing schedule (months 0, 2, and 6) and/or given before, or concomitantly with, a vaccine against Japanese encephalitis (JE). RESULTS: Based on dengue virus serotype-specific neutralizing antibody (NAb), the accelerated dosing schedule was comparable to the 0, 6, and 12-month schedule. Giving JE vaccine concurrently with CYD-TDV did not result in an increase in overall NAb titers. Immunophenotyping of peripheral blood mononuclear cells revealed an increase in activated CD8+ T cells after CYD-TDV vaccination, a phenomenon that was greatest for the JE vaccine primed. CONCLUSIONS: We conclude that an accelerated dosing schedule of CYD-TDV results in essentially equivalent dengue serotype-specific NAb titers as the currently used schedule, and there may be an early benefit in antibody titers and activated CD8+ T cells by the administration of the JE vaccine before CYD-TDV vaccination.

Multiplexed FluoroSpot for the Analysis of Dengue Virus- and Zika Virus-Specific and Cross-Reactive Memory B Cells.

Dengue virus (DENV) and Zika virus (ZIKV) are mosquito-borne pathogens that have a significant impact on human health. Immune sera, mAbs, and memory B cells (MBCs) isolated from patients infected with one DENV type can be cross-reactive with the other three DENV serotypes and even more distantly related flaviviruses such as ZIKV. Conventional ELISPOTs effectively measure Ab-secreting B cells but because they are limited to the assessment of a single Ag at a time, it is challenging to distinguish serotype-specific and cross-reactive MBCs in the same well. We developed a novel multifunction FluoroSpot assay using fluorescently labeled DENV and ZIKV (FLVs) that measures the cross-reactivity of Abs secreted by single B cells. Conjugation efficiency and recognition of FLVs by virus-specific Abs were confirmed by flow cytometry. Using a panel of DENV immune, ZIKV immune, and naive PBMC, FLVs were able to simultaneously detect DENV serotype-specific, ZIKV-specific, DENV serotype cross-reactive, and DENV/ZIKV cross-reactive Abs secreted by individual MBCs. Our findings indicate that the FLVs are sensitive and specific tools to detect specific and cross-reactive MBCs. These reagents will allow the assessment of the breadth as well as the durability of DENV/ZIKV B cell responses following vaccination or natural infection. This novel approach using FLVs in a FluoroSpot assay can be applied to other diseases such as influenza in which prior immunity with homosubtype- or heterosubtype-specific MBCs may influence subsequent infections.

Impact of prior flavivirus immunity on Zika virus infection in rhesus macaques.

Studies have demonstrated cross-reactivity of anti-dengue virus (DENV) antibodies in human sera against Zika virus (ZIKV), promoting increased ZIKV infection in vitro. However, the correlation between in vitro and in vivo findings is not well characterized. Thus, we evaluated the impact of heterotypic flavivirus immunity on ZIKV titers in biofluids of rhesus macaques. Animals previously infected (≥420 days) with DENV2, DENV4, or yellow fever virus were compared to flavivirus-naïve animals following infection with a Brazilian ZIKV strain. Sera from DENV-immune macaques demonstrated cross-reactivity with ZIKV by antibody-binding and neutralization assays prior to ZIKV infection, and promoted increased ZIKV infection in cell culture assays. Despite these findings, no significant differences between flavivirus-naïve and immune animals were observed in viral titers, neutralizing antibody levels, or immune cell kinetics following ZIKV infection. These results indicate that prior infection with heterologous flaviviruses neither conferred protection nor increased observed ZIKV titers in this non-human primate ZIKV infection model.

Dynamics of Dengue Virus (DENV)-Specific B Cells in the Response to DENV Serotype 1 Infections, Using Flow Cytometry With Labeled Virions.

BACKGROUND: The development of reagents to identify and characterize antigen-specific B cells has been challenging. METHODS: We recently developed Alexa Fluor-labeled dengue viruses (AF DENVs) to characterize antigen-specific B cells in the peripheral blood of DENV-immune individuals. RESULTS: In this study, we used AF DENV serotype 1 (AF DENV-1) together with AF DENV-2 on peripheral blood mononuclear cells (PBMCs) from children in Thailand with acute primary or secondary DENV-1 infections to analyze the phenotypes of antigen-specific B cells that reflected their exposure or clinical diagnosis. DENV serotype-specific and cross-reactive B cells were identified in PBMCs from all subjects. Frequencies of AF DENV(+) class-switched memory B cells (IgD(-)CD27(+) CD19(+) cells) reached up to 8% during acute infection and early convalescence. AF DENV-labeled B cells expressed high levels of CD27 and CD38 during acute infection, characteristic of plasmablasts, and transitioned into memory B cells (CD38(-)CD27(+)) at the early convalescent time point. There was higher activation of memory B cells early during acute secondary infection, suggesting reactivation from a previous DENV infection. CONCLUSIONS: AF DENVs reveal changes in the phenotype of DENV serotype-specific and cross-reactive B cells during and after natural DENV infection and could be useful in analysis of the response to DENV vaccination.

Low-dose dengue virus 3 human challenge model: a phase 1 open-label study.

Dengue human infection models present an opportunity to explore the potential of a vaccine, anti-viral or immuno-compound for clinical benefit in a controlled setting. Here we report the outcome of a phase 1 open-label assessment of a low-dose dengue virus 3 (DENV-3) challenge model (NCT04298138), in which nine participants received a subcutaneous inoculation with 0.5 ml of a 1.4 × 103 plaque-forming unit per ml suspension of the attenuated DENV-3 strain CH53489. The primary and secondary endpoints of the study were to assess the safety of this DENV-3 strain in healthy flavivirus-seronegative individuals. All participants developed RNAaemia within 7 days after inoculation with peak titre ranging from 3.13 × 104 to 7.02 × 108 genome equivalents per ml. Solicited symptoms such as fever and rash, clinical laboratory abnormalities such as lymphopenia and thrombocytopenia, and self-reported symptoms such as myalgia were consistent with mild-to-moderate dengue in all volunteers. DENV-3-specific seroconversion and memory T cell responses were observed within 14 days after inoculation as assessed by enzyme-linked immunosorbent assay and interferon-gamma-based enzyme-linked immunospot. RNA sequencing and serum cytokine analysis revealed anti-viral responses that overlapped with the period of viraemia. The magnitude and frequency of clinical and immunologic endpoints correlated with an individual's peak viral titre.

Efficacy and immunogenicity following dengue virus-1 human challenge after a tetravalent prime-boost dengue vaccine regimen: an open-label, phase 1 trial.

BACKGROUND: Dengue human infection models (DHIMs) are important tools to down-select dengue vaccine candidates and establish tetravalent efficacy before advanced clinical field trials. We aimed to provide data for the safety and immunogenicity of DHIM and evaluate dengue vaccine efficacy. METHODS: We performed an open-label, phase 1 trial at the University of Maryland (Baltimore, MD, USA). Eligible participants were healthy individuals aged 18-50 years who either previously received a tetravalent dengue purified inactivated vaccine prime followed by a live-attenuated vaccine boost (ie, the vaccinee group), or were unvaccinated flavivirus-naive participants (ie, the control group). Participants in the vaccinee group with detectable pre-challenge dengue virus-1 neutralising antibody titres and flavivirus-naive participants in the control group were inoculated with dengue virus-1 strain 45AZ5 in the deltoid region, 27-65 months following booster dosing. These participants were followed-up from days 4-16 following dengue virus-1 live virus human challenge, with daily real-time quantitative PCR specific to dengue virus-1 RNA detection, and dengue virus-1 solicited local and systemic adverse events were recorded. The primary outcomes were safety (ie, solicited local and systemic adverse events) and vaccine efficacy (ie, dengue virus-1 RNAaemia) following dengue challenge. This study is registered with ClinicalTrials.gov, number NCT04786457. FINDINGS: In January 2021, ten eligible participants were enrolled; of whom, six (60%) were in the vaccinee group and four (40%) were in the control group. Daily quantitative PCR detected dengue virus-1 RNA in nine (90%) of ten participants (five [83%] of six in the vaccinee group and all four [100%] in the control group). The mean onset of RNAaemia occurred on day 5 (SD 1·0) in the vaccinee group versus day 8 (1·5) in the control group (95% CI 1·1-4·9; p=0·007), with a trend towards reduced RNAaemia duration in the vaccinee group compared with the control group (8·2 days vs 10·5 days; 95% CI -0·08 to 4·68; p=0·056). Mild-to-moderate symptoms (nine [90%] of ten), leukopenia (eight [89%] of nine), and elevated aminotransferases (seven [78%] of nine) were commonly observed. Severe adverse events were detected only in the vaccinee group (fever ≥38·9°C in three [50%] of six, headache in one [17%], and transient grade 4 aspartate aminotransferase elevation in one [17%]). No deaths were reported. INTERPRETATION: Participants who had tetravalent dengue purified inactivated vaccine prime and live-attenuated vaccine boost were unprotected against dengue virus-1 infection and further showed increased clinical, immunological, and transcriptomic evidence for inflammation potentially mediated by pre-existing infection-enhancing antibodies. This study highlights the impact of small cohort, human challenge models studying dengue pathogenesis and downstream vaccine development. FUNDING: Military Infectious Disease Research Program and Medical Technology Enterprise Consortium and Advanced Technology International.

mRNA-1273 vaccination protects against SARS-CoV-2-elicited lung inflammation in nonhuman primates.

Vaccine-elicited SARS-CoV-2 antibody responses are an established correlate of protection against viral infection in humans and nonhuman primates. However, it is less clear that vaccine-induced immunity is able to limit infection-elicited inflammation in the lower respiratory tract. To assess this, we collected bronchoalveolar lavage fluid samples after SARS-CoV-2 strain USA-WA1/2020 challenge from rhesus macaques vaccinated with mRNA-1273 in a dose-reduction study. Single-cell transcriptomic profiling revealed a broad cellular landscape 48 hours after challenge, with distinct inflammatory signatures that correlated with viral RNA burden in the lower respiratory tract. These inflammatory signatures included phagocyte-restricted expression of chemokines, such as CXCL10 and CCL3, and the broad expression of IFN-induced genes, such as MX1, ISG15, and IFIT1. Induction of these inflammatory profiles was suppressed by prior mRNA-1273 vaccination in a dose-dependent manner and negatively correlated with prechallenge serum and lung antibody titers against SARS-CoV-2 spike. These observations were replicated and validated in a second independent macaque challenge study using the B.1.351/Beta variant of SARS-CoV-2. These data support a model wherein vaccine-elicited antibody responses restrict viral replication following SARS-CoV-2 exposure, including limiting viral dissemination to the lower respiratory tract and infection-mediated inflammation and pathogenesis.

Characterization of B-cell and T-cell responses to a tetravalent dengue purified inactivated vaccine in healthy adults.

The increasing global impact of dengue underscores the need for a dengue virus (DENV) vaccine. We assessed B-cell and T-cell responses following vaccination with four formulations of a tetravalent dengue purified inactivated vaccine (DPIV) in dengue-primed and dengue-naive adults from two studies (NCT01666652, NCT01702857). Frequencies of DPIV-induced memory B cells specific to each DENV serotype remained high up to 12 months post-vaccination, and were higher in the dengue-primed than dengue-naive adults. A subsequent DPIV booster dose induced strong anamnestic B-cell responses. Multifunctional CD4+ T cells (predominantly expressing IL-2) were induced by DPIV, with higher frequencies in dengue-primed adults. DPIV-induced CD4+ T cells also demonstrated in vitro proliferative capacity and antigen-specific production of GM-CSF, IFN-γ, and IL-13. CD8+ T-cell responses were undetectable in dengue-naive adults and low in dengue-primed individuals. B- and T-cell responses persisted up to 12 months post-vaccination in both dengue-primed and dengue-naive adults.

Memory CD8+ T cells from naturally acquired primary dengue virus infection are highly cross-reactive.

Cross-reactive memory T cells induced by primary infection with one of the four serotypes of dengue virus (DENV) are hypothesized to have an immunopathological function in secondary heterologous DENV infection. To define the T-cell response to heterologous serotypes, we isolated HLA-A(*)1101-restricted epitope-specific CD8(+) T-cell lines from primary DENV-immune donors. Cell lines exhibited marked cross-reactivity toward peptide variants representing the four DENV serotypes in tetramer binding and functional assays. Many clones responded similarly to homologous and heterologous serotypes with striking cross-reactivity between the DENV-1 and DENV-3 epitope variants. In vitro-stimulated T-cell lines consistently revealed a hierarchical induction of MIP-1ß>degranulation>tumor necrosis factor α (TNFα)>interferon-γ (IFNγ), which depended on the concentration of agonistic peptide. Phosphoflow assays showed peptide dose-dependent phosphorylation of ERK1/2, which correlated with cytolysis, degranulation, and induction of TNFα and IFNγ, but not MIP-1ß production. This is the first study to show significant DENV serotype-cross-reactivity of CD8(+) T cells after naturally acquired primary infection. We also show qualitatively different T-cell receptor signaling after stimulation with homologous and heterologous peptides. Our data support a model whereby the order of sequential DENV infections influences the immune response to secondary heterologous DENV infection, contributing to varying disease outcomes.

Stochastic Model of the Adaptive Immune Response Predicts Disease Severity and Captures Enhanced Cross-Reactivity in Natural Dengue Infections.

The dengue virus circulates as four distinct serotypes, where a single serotype infection is typically asymptomatic and leads to acquired immunity against that serotype. However, the developed immunity to one serotype is thought to underlie the severe manifestation of the disease observed in subsequent infections from a different serotype. We developed a stochastic model of the adaptive immune response to dengue infections. We first delineated the mechanisms initiating and sustaining adaptive immune responses during primary infections. We then contrasted these immune responses during secondary infections of either a homotypic or heterotypic serotype to understand the role of pre-existing and reactivated immune pathways on disease severity. Comparison of non-symptomatic and severe cases from heterotypic infections demonstrated that overproduction of specific antibodies during primary infection induces an enhanced population of cross-reactive antibodies during secondary infection, ultimately leading to severe disease manifestations. In addition, the level of disease severity was found to correlate with immune response kinetics, which was dependent on beginning lymphocyte levels. Our results detail the contribution of specific lymphocytes and antibodies to immunity and memory recall that lead to either protective or pathological outcomes, allowing for the understanding and determination of mechanisms of protective immunity.

Persistent COVID-19 Symptoms Minimally Impact the Development of SARS-CoV-2-Specific T Cell Immunity.

SARS-CoV-2 represents an unprecedented public health challenge. While the majority of SARS-CoV-2-infected individuals with mild-to-moderate COVID-19 resolve their infection with few complications, some individuals experience prolonged symptoms lasting for weeks after initial diagnosis. Persistent viral infections are commonly accompanied by immunologic dysregulation, but it is unclear if persistent COVID-19 impacts the development of virus-specific cellular immunity. To this end, we analyzed SARS-CoV-2-specific cellular immunity in convalescent COVID-19 patients who experienced eight days or fewer of COVID-19 symptoms or symptoms persisting for 18 days or more. We observed that persistent COVID-19 symptoms were not associated with the development of an overtly dysregulated cellular immune response. Furthermore, we observed that reactivity against the N protein from SARS-CoV-2 correlates with the amount of reactivity against the seasonal human coronaviruses 229E and NL63. These results provide insight into the processes that regulate the development of cellular immunity against SARS-CoV-2 and related human coronaviruses.

Monomeric IgA Antagonizes IgG-Mediated Enhancement of DENV Infection.

Dengue virus (DENV) is a prevalent human pathogen, infecting approximately 400 million individuals per year and causing symptomatic disease in approximately 100 million. A distinct feature of dengue is the increased risk for severe disease in some individuals with preexisting DENV-specific immunity. One proposed mechanism for this phenomenon is antibody-dependent enhancement (ADE), in which poorly-neutralizing IgG antibodies from a prior infection opsonize DENV to increase infection of Fc gamma receptor-bearing cells. While IgM and IgG are the most commonly studied DENV-reactive antibody isotypes, our group and others have described the induction of DENV-specific serum IgA responses during dengue. We hypothesized that monomeric IgA would be able to neutralize DENV without the possibility of ADE. To test this, we synthesized IgG and IgA versions of two different DENV-reactive monoclonal antibodies. We demonstrate that isotype-switching does not affect the antigen binding and neutralization properties of the two mAbs. We show that DENV-reactive IgG, but not IgA, mediates ADE in Fc gamma receptor-positive K562 cells. Furthermore, we show that IgA potently antagonizes the ADE activity of IgG. These results suggest that levels of DENV-reactive IgA induced by DENV infection might regulate the overall IgG mediated ADE activity of DENV-immune plasma in vivo, and may serve as a predictor of disease risk.

Longitudinal Analysis of Dengue Virus-Specific Memory T Cell Responses and Their Association With Clinical Outcome in Subsequent DENV Infection.

Memory T cells resulting from primary dengue virus (DENV) infection are hypothesized to influence the clinical outcome of subsequent DENV infection. However, the few studies involving prospectively collected blood samples have found weak and inconsistent associations with outcome and variable temporal trends in DENV-specific memory T cell responses between subjects. This study used both ex-vivo and cultured ELISPOT assays to further evaluate the associations between DENV serotype-cross-reactive memory T cells and severity of secondary infection. Using ex-vivo ELISPOT assays, frequencies of memory T cells secreting IFN-γ in response to DENV structural and non-structural peptide pools were low in PBMC from multiple time points prior to symptomatic secondary DENV infection and showed a variable response to infection. There were no differences in responses between subjects who were not hospitalized (NH, n=6) and those who were hospitalized with dengue hemorrhagic fever (hDHF, n=4). In contrast, responses in cultured ELISPOT assays were more reliably detectable prior to secondary infection and showed more consistent increases after infection. Responses in cultured ELISPOT assays were higher in individuals with hDHF (n=8) compared to NH (n=9) individuals before the secondary infection, with no difference between these groups after infection. These data demonstrate an association of pre-existing DENV-specific memory responses with the severity of illness in subsequent DENV infection, and suggest that frequencies of DENV-reactive T cells measured after short-term culture may be of particular importance for assessing the risk for more severe dengue disease.

Enhanced dengue vaccine virus replication and neutralizing antibody responses in immune primed rhesus macaques.

Antibody-dependent enhancement (ADE) is suspected to influence dengue virus (DENV) infection, but the role ADE plays in vaccination strategies incorporating live attenuated virus components is less clear. Using a heterologous prime-boost strategy in rhesus macaques, we examine the effect of priming with DENV purified inactivated vaccines (PIVs) on a tetravalent live attenuated vaccine (LAV). Sera exhibited low-level neutralizing antibodies (NAb) post PIV priming, yet moderate to high in vitro ADE activity. Following LAV administration, the PIV primed groups exhibited DENV-2 LAV peak viremias up to 1,176-fold higher than the mock primed group, and peak viremia correlated with in vitro ADE. Furthermore, PIV primed groups had more balanced and higher DENV-1-4 NAb seroconversion and titers than the mock primed group following LAV administration. These results have implications for the development of effective DENV vaccine prime-boost strategies and for our understanding of the role played by ADE in modulating DENV replication.