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Patients receiving CD19 CAR T-cell therapy for relapsed/refractory lymphoma experience prolonged and profound B-cell aplasia and hypogammaglobulinemia, placing them at a higher risk for severe COVID-19. Independently, Oh et al and Atanackovic et al demonstrate that despite attenuated humoral response to mRNA-based vaccines, patients demonstrate normal or heightened functional T-cell responses, including antiviral T-cell activity against SARS-CoV-2 variants including Omicron. Collectively, these data reinforce the importance of COVID-19 vaccination following CD19 CAR T-cell therapy, despite long-term B-cell aplasia.

A self-transcribing and replicating RNA (STARR)-based vaccine (LUNAR-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full-length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolongs SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell-mediated immunity produced a strong viral antigen-specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for interferon (IFN)γ and interleukin-4 (IL-4)-positive CD4+ T helper (Th) lymphocytes as well as anti-spike glycoprotein immunoglobulin G (IgG)2a/IgG1 ratios supported a strong Th1-dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of LUNAR-COV19 as a single-dose vaccine.

Purpose of Review At the turn of the nineteenth century, yellow fever (YF) was considered the most dangerous infectious disease with high case fatality. Subsequent, mass vaccination campaigns coupled with widespread elimination of the YF mosquito vector significantly decreased YF cases and reduced outbreaks to the tropical and subtropical forested regions of Africa and South America. Recent Findings However, recent (2016) large outbreaks in Angola, Democratic Republic of Congo (DRC), and South-Eastern Brazil, where previously had been demarcated as low-risk regions, have highlighted the possibility of a rapidly changing epidemiology and the potential re-emergence of yellow fever virus (YFV). Furthermore, the first-ever importation of YFV into Asia has highlighted the potential fear of YFV emerging as a global threat. Summary In this review, we describe the changing epidemiology of YF outbreaks and highlight the use of public health policies, therapeutics, and vaccination as tools to help eliminate future YFV outbreaks.

Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. This vaccine platform has been extended to encode an alphavirus replicase that self-amplifies the full length mRNA and SARS-CoV-2 spike (S) transgene (self-amplifying mRNA or sa mRNA). However, early phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual anti-S T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2. Correlation with live attenuated vaccines was weaker. Our findings suggest the potential for sa-mRNA to be further developed to be among the most potent forms of vaccines in our arsenal to prevent infectious diseases.

Background Protection offered by coronavirus disease 2019 (COVID-19) vaccines wanes over time, requiring an evaluation of different boosting strategies to revert such a trend and enhance the quantity and quality of Spike-specific humoral and cellular immune responses. These immunological parameters in homologous or heterologous vaccination boosts have thus far been studied for mRNA and ChAdOx1 nCoV-19 vaccines, but knowledge on individuals who received a single dose of Ad26.COV2.S is lacking. Methods We studied Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals (n = 55) who were either primed with Ad26.COV2.S only (n = 13) or were boosted with a homologous (Ad26.COV2.S, n = 28) or heterologous (BNT162b2, n = 14) second dose. We compared our findings with the results found in individuals vaccinated with a single (n = 16) or double (n = 44) dose of BNT162b2. Findings We observed that a strategy of heterologous vaccination enhanced the quantity and breadth of both Spike-specific humoral and cellular immunity in Ad26.COV2.S-vaccinated individuals. In contrast, the impact of the homologous boost was quantitatively minimal in Ad26.COV2.S-vaccinated individuals, and Spike-specific antibodies and T cells were narrowly focused to the S1 region. Conclusions Despite the small sample size of the study and the lack of well-defined correlates of protection against COVID-19, the immunological features detected support the utilization of a heterologous vaccine boost in individuals who received Ad26.COV2.S vaccination. Funding This study is partially supported by the Singapore Ministry of Health’s National Medical Research Council under its COVID-19 Research Fund (COVID19RF3-0060, COVID19RF-001, and COVID19RF-008), The Medical College St. Bartholomew’s Hospital Trustees – Pump Priming Fund for SMD COVID-19 Research., Graphical abstract, Context and significance Protection offered by COVID-19 vaccines wanes over time. We evaluated whether individuals vaccinated with a single dose of Johnson & Johnson’s vaccine Ad26.COV2.S would benefit from a second dose of Ad26.COV2.S (homologous boost) or of the Pfizer-BioNTech mRNA vaccine BNT162b2 (heterologous boost). The study of different vaccination regimens is critical to optimize future vaccine strategies. We analyzed the quantity and quality of antibodies and T cells specific for Spike, demonstrating that Spike-specific antibodies and T cells were augmented preferentially following a boost with BNT162b2. A heterologous boost also expanded the ability of antibodies and T cells to recognize different sections of Spike. These data support the use of a heterologous mix-and-match strategy in individuals vaccinated with a single dose of Ad26.COV2.S., Homologous versus heterologous boosting strategies were evaluated in single-dose Ad26.COV2.S vaccines. Magnitude of Spike-specific humoral and cellular immunity was augmented following a boost with BNT162b2. Heterologous vaccination expanded the ability of both humoral and cellular immunity to recognize multiple regions of Spike. Convalescent individuals responded equally to both boosting strategies.

Protection offered by COVID-19 vaccines wanes over time, requiring an evaluation of different boosting strategies to revert such a trend and enhance the quantity and quality of Spike-specific humoral and cellular immune responses. These immunological parameters in homologous or heterologous vaccination boosts have thus far been studied for mRNA and ChAdOx1 nCoV-19 vaccines, but knowledge on individuals who received a single dose of Ad26.COV2.S is lacking.We studied Spike-specific humoral and cellular immunity in Ad26.COV2.S vaccinated individuals (n=55) who were either primed with Ad26.COV2.S only (n=13), or boosted with a homologous (Ad26.COV2.S, n=28) or heterologous (BNT162b2, n=14) second dose. We compared our findings with the results found in individuals vaccinated with a single (n=16) or double (n=44) dose of BNT162b2. We observed that a strategy of heterologous vaccination enhanced the quantity and breadth of both, Spike-specific humoral and cellular immunity in Ad26.COV2.S vaccinated. In contrast, the impact of homologous boost was quantitatively minimal in Ad26.COV2.S vaccinated and Spike-specific antibodies and T cells were narrowly focused to the S1 region. Although a direct association between quantity and quality of immunological parameters and in vivo protection has not been demonstrated, the immunological features of Spike-specific humoral and cellular immune responses support the utilization of a heterologous strategy of vaccine boost in individuals who received Ad26.COV2.S vaccination.

BackgroundThe pandemic of coronavirus disease-19 (Covid-19) continues to afflict the lives and livelihoods of many as global demand for vaccine supply remains unmet.MethodsPhase 1 of this trial (N=42) assessed the safety, tolerability and immunogenicity of ascending levels of one-dose ARCT-021, a self-amplifying mRNA vaccine against Covid-19. Phase 2 (N=64) tested two-doses of ARCT-021 given 28 days apart. Both young and older adults were enrolled. The primary safety outcomes were local and systemic solicited adverse events (AEs) reported immediately and up to 7 days post-inoculation and unsolicited events reported up to 56 days after inoculation. Secondary and exploratory outcomes were antibody and T cell responses to vaccination, respectively.ResultsARCT-021 was well tolerated up to one 7.5 μg dose and two 5.0 μg doses. Local solicited AEs, namely injection-site pain and tenderness, as well as systemic solicited AEs, such as fatigue, headache and myalgia, were more common in ARCT-021 than placebo recipients, and in younger than older adults. Seroconversion rate for anti-S IgG was 100% in all cohorts except for the 1 μg one-dose in younger adults and the 7.5 μg one-dose in older adults, which were each 80%. Neutralizing antibody titers increased with increasing dose although the responses following 5.0 μg and 7.5 μg ARCT-021 were similar. Anti-S IgG titers overlapped with those in Covid-19 convalescent plasma. ARCT-021 also elicited T-cell responses against the S glycoprotein.ConclusionTaken collectively, the favorable safety and immunogenicity profiles support further clinical development of ARCT-021.

Background RNA vaccines against coronavirus disease 2019 (COVID-19) have demonstrated ∼95% efficacy in phase III clinical trials. Although complete vaccination consisted of 2 doses, the onset of protection for both licensed RNA vaccines was observed as early as 12 days after a single dose. The adaptive immune response that coincides with this onset of protection could represent the necessary elements of immunity against COVID-19. Methods Serological and T cell analysis was performed in a cohort of 20 healthcare workers after receiving the first dose of the Pfizer/BioNTech BNT162b2 vaccine. The primary endpoint was the adaptive immune responses detectable at days 7 and 10 after dosing. Findings Spike-specific T cells and binding antibodies were detectable 10 days after the first dose of the vaccine, in contrast to receptor-blocking and severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) neutralizing antibodies, which were mostly undetectable at this early time point. Conclusions Our findings suggest that early T cell and binding antibody responses, rather than either receptor-blocking or virus neutralizing activity, induced early protection against COVID-19. Funding The study was funded by a generous donation from The Hour Glass to support COVID-19 research., Graphical abstract, Context and significance RNA vaccines have shown efficacy in preventing coronavirus disease 2019 (COVID-19) as early as 12 days after the first dose. Vaccine efficacy onset presents a unique opportunity to define the necessary elements of immunity against COVID-19. Kalimuddin et al. tracked the serological and T cell responses longitudinally in 20 healthcare workers after the first Pfizer/BioNTech BNT162b2 vaccine dose. Anti-spike immunoglobulin G (IgG) and IgA antibodies and spike-specific T cells were detectable at day 10 after the first dose; neutralizing and receptor-blocking antibodies remained mostly undetectable at this time point. These results suggest that binding antibodies and T cell responses are responsible for early protection against COVID-19 and call for circumspection on the prevailing notion that neutralizing antibodies are absolutely required for protection., Kalimuddin et al. show that spike protein-binding antibodies and spike-specific T cells, but not neutralizing antibodies, could explain the onset of vaccine efficacy after the first dose of BNT162b2 vaccination. Their findings provide insight into the components of the adaptive immune response necessary for protection against COVID-19.

Additional file 3: Table S2. Identified protein within subsets of interests during chromosomal genome comparison between S. sonnei, S. flexneri, pathogenic and non-pathogenic E. coli.

Additional file 4: Supplementary file 2. Script used to retrieve annotation and protein sequences from the input Shigella genomes.

Additional file 2: Table S1. List of Shigella and E. coli strains used for the bioinformatics genome comparison.

Additional file 9: Figure S3. Convalescent antibody responses to top reactive Shigella antigens by age and sex.

Additional file 6: Table S4. The recombinant expression of highly reactive Shigella proteins for in vivo immunogen testing.

RNA vaccines against Covid-19 have demonstrated ~95% efficacy in Phase III clinical trials. Although complete vaccination consisted of two-doses, the onset of protection for both licensed RNA vaccines was observed as early as 12 days after a single dose. The adaptive immune response that coincides with this onset of protection could represent the necessary elements of immunity against Covid-19. Herein, we tracked the early adaptive immune responses after Covid-19 RNA vaccination, in a cohort of 20 healthcare workers. Our findings suggest that early T cell and binding antibody responses, rather than either receptor-blocking or virus neutralizing activity, induced early protection against Covid-19. Funding: This study was partially funded through a generousdonation from The Hourglass to support Covid-19 research in ViREMiCS. SK receives salary support from the Transition Award, RdA receives funding from the Open Research Fund Young Investigator Award, JGL and EEO receive salary support from the Clinician Scientist Award, and AB receives salary support from the Singapore Translational Research Award, all administered by the National Medical Research Council of Singapore. Conflict of Interest: Duke-NUS Medical School is in partnership with Arcturus Therapeutics to develop a self-replicating RNA vaccine against Covid-19, with EEO as the principal investigator. No monetary or personal benefits are derived from this partnership. Ethical Approval: This study was approved by the SingHealth Centralized Institutional Review Board (CIRB/F2021/2014). Healthcare workers (HCWs) from the Singapore Health Services institutions whowere eligible for Covid-19 vaccination were invited to participate in this study, and written informed consent was obtained.

Additional file 1: Supplementary file 1. In-house script to retrieve complete Shigella and Escherichia coli chromosomal sequences from GenBank (accessed July 2014).

Additional file 7: Figure S1. No statistically significant seroconversion to Shigella antigens detected in diarrheal patients during Salmonella infections.

Additional file 8: Figure S2. Antibody responses to natural Shigella infections are influenced by severity of disease.

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

A self-transcribing and replicating RNA (STARR™) based vaccine (LUNAR®-COV19) has been developed to prevent SARS-CoV-2 infection. The vaccine encodes an alphavirus-based replicon and the SARS-CoV-2 full length spike glycoprotein. Translation of the replicon produces a replicase complex that amplifies and prolong SARS-CoV-2 spike glycoprotein expression. A single prime vaccination in mice led to robust antibody responses, with neutralizing antibody titers increasing up to day 60. Activation of cell mediated immunity produced a strong viral antigen specific CD8+ T lymphocyte response. Assaying for intracellular cytokine staining for IFN-γ and IL-4 positive CD4+ T helper lymphocytes as well as anti-spike glycoprotein IgG2a/IgG1 ratios supported a strong Th1 dominant immune response. Finally, single LUNAR-COV19 vaccination at both 2 μg and 10 μg doses completely protected human ACE2 transgenic mice from both mortality and even measurable infection following wild-type SARS-CoV-2 challenge. Our findings collectively suggest the potential of Lunar-COV19 as a single dose vaccine.

Antibodies that lack specific sugar modifications can worsen acute viral diseases

Additional file 2: Supplementary table 1. Clinical and laboratory characteristics of patients with DHF and DF. Supplementary table 2. Definitions of terms with regard to DENV infections.

Additional file 1: Supplementary figure 1. The hierarchical gating strategy used to identify Tfh cells and to assess their phenotype and functionality. Supplementary figure 2. The frequency of Tfh cells and IL-21 producing Tfh cells in patients with acute dengue during acute phase and convalescent phase. Supplementary figure 3. The hierarchical gating strategy used to identify plasmablasts. Cells were first gated on the PBMCS, then the singlets were identified by gating on FSC-height and area. These cells were then gated on the live cells and subsequently on CD19, CD27 and CD38. Supplementary figure 4. The frequency of plasmablasts in a patient with acute dengue in the acute and convalescent phase.

Dengue disease is a large public health problem that mainly afflicts tropical and subtropical regions. Understanding of the correlates of protection against dengue virus (DENV) is poor and hinders the development of a successful human vaccine. The present study aims to define DENV-specific CD8 + T cell responses in general and those of HLA alleles associated with dominant responses in particular. In human blood donors in Nicaragua, we observed a striking dominance of HLA B-restricted responses in general and of the allele B*35:01 in particular. Comparing these patterns to those in the general population of Sri Lanka, we found a strong correlation between restriction of the HLA allele and the breadth and magnitude of CD8 + T cell responses, suggesting that HLA genes profoundly influence the nature of responses. The majority of gamma interferon (IFN-γ) responses were associated with effector memory phenotypes, which were also detected in non-B*35:01-expressing T cells. However, only the B*35:01 DENV-specific T cells were associated with marked expression of the programmed death 1 protein (PD-1). These cells did not coexpress other inhibitory receptors and were able to proliferate in response to DENV-specific stimulation. Thus, the expression of particular HLA class I alleles is a defining characteristic influencing the magnitude and breadth of CD8 responses, and a distinct, highly differentiated phenotype is specifically associated with dominant CD8 + T cells. These results are of relevance for both vaccine design and the identification of robust correlates of protection in natural immunity. IMPORTANCE Dengue is an increasingly significant public health problem as its mosquito vectors spread over greater areas; no vaccines against the virus have yet been approved. An important step toward vaccine development is defining protective immune responses; toward that end, we here characterize the phenotype of the immunodominant T cell responses. These DENV-reactive T cells express high levels of the receptor programmed death 1 protein (PD-1), while those from disease-susceptible alleles do not. Not only does this represent a possible correlate of immunodominance, but it raises the hypothesis that PD-1 might be a regulator that prevents excessive damage while preserving antiviral function. Further, as this study employs distinct populations (Nicaraguan and Sri Lankan donors), we also confirmed that this pattern holds despite geographic and ethnic differences. This finding indicates that HLA type is the major determinant in shaping T cell responses.

Vaccination is an effective approach to reduce disease burden. High vaccination coverage blocks pathogen transmission to ensure herd immunity. However, the concept of herd immunity assumes that vaccinated individuals cannot be infected and mediate silent pathogen transmission. While the correlates of vaccine-mediated protection against disease have been examined, the correlates of sterilizing immunity that prevents infection have not been systematically defined. Here, we used full genome expression profiling to explore the molecular correlates of serological response and non-response to measles, mumps and rubella (MMR) vaccination as surrogates of infection and sterilizing immunity, respectively. We observed that the antibody titers needed to sterilize infection with the vaccine strains were higher than current WHO disease protection thresholds. In subjects with baseline antibodies below such sterilizing immunity thresholds, serological non-response to MMR vaccination was associated with gene expression profile indicative of early T-cell activation and signalling. Specifically, genes that regulate T-cell function and response were induced at day 1 post-vaccination in non-responders but not in responders. These findings suggest that rapid T-cell response prevented MMR vaccine infection to limit antigenic presentation and hence serological response. Collectively, our findings suggest an important role for T-cells in engendering sterilizing immunity. publishedVersion

We found a high transfer ratio of anti-nontyphoidal Salmonella (NTS) antibodies from mothers to infants. Furthermore, despite a high seroincidence of NTS in infants, maternally acquired antibodies provided protection from seroconversion. Therefore, we propose prenatal immunization against NTS as a possible strategy for protecting infants from NTS disease., Background Nontyphoidal Salmonella (NTS) organisms are a major cause of gastroenteritis and bacteremia, but little is known about maternally acquired immunity and natural exposure in infant populations residing in areas where NTS disease is highly endemic. Methods We recruited 503 pregnant mothers and their infants (following delivery) from urban areas in Vietnam and followed infants until they were 1 year old. Exposure to the dominant NTS serovars, Salmonella enterica serovars Typhimurium and Enteritidis, were assessed using lipopolysaccharide (LPS) O antigen–specific antibodies. Antibody dynamics, the role of maternally acquired antibodies, and NTS seroincidence rates were modeled using multivariate linear risk factor models and generalized additive mixed-effect models. Results Transplacental transfer of NTS LPS–specific maternal antibodies to infants was highly efficient. Waning of transplacentally acquired NTS LPS–specific antibodies at 4 months of age left infants susceptible to Salmonella organisms, after which they began to seroconvert. High seroincidences of S. Typhimurium and S. Enteritidis LPS were observed, and infants born with higher anti-LPS titers had greater plasma bactericidal activity and longer protection from seroconversion. Conclusions Although Vietnamese infants have extensive exposure to NTS, maternally acquired antibodies appear to play a protective role against NTS infections during early infancy. These findings suggest that prenatal immunization may be an appropriate strategy to protect vulnerable infants from NTS disease.

We identified high antimicrobial usage and high antimicrobial resistance in children hospitalized with bloody and/or mucoid bacteria-associated diarrhea. There was no clinical benefit of antimicrobial therapy in children with diarrhea; adequately powered randomized controlled trials are required., Background Pediatric diarrheal disease presents a major public health burden in low- to middle-income countries. The clinical benefits of empirical antimicrobial treatment for diarrhea are unclear in settings that lack reliable diagnostics and have high antimicrobial resistance (AMR). Methods We conducted a prospective multicenter cross-sectional study of pediatric patients hospitalized with diarrhea containing blood and/or mucus in Ho Chi Minh City, Vietnam. Clinical parameters, including disease outcome and treatment, were measured. Shigella, nontyphoidal Salmonella (NTS), and Campylobacter were isolated from fecal samples, and their antimicrobial susceptibility profiles were determined. Statistical analyses, comprising log-rank tests and accelerated failure time models, were performed to assess the effect of antimicrobials on disease outcome. Results Among 3166 recruited participants (median age 10 months; interquartile range, 6.5–16.7 months), one-third (1096 of 3166) had bloody diarrhea, and 25% (793 of 3166) were culture positive for Shigella, NTS, or Campylobacter. More than 85% of patients (2697 of 3166) were treated with antimicrobials; fluoroquinolones were the most commonly administered antimicrobials. AMR was highly prevalent among the isolated bacteria, including resistance against fluoroquinolones and third-generation cephalosporins. Antimicrobial treatment and multidrug resistance status of the infecting pathogens were found to have no significant effect on outcome. Antimicrobial treatment was significantly associated with an increase in the duration of hospitalization with particular groups of diarrheal diseases. Conclusions In a setting with high antimicrobial usage and high AMR, our results imply a lack of clinical benefit for treating diarrhea with antimicrobials; adequately powered randomized controlled trials are required to assess the role of antimicrobials for diarrhea.

Natural dengue virus (DENV) infection in humans induces antibodies (Abs) that neutralize the serotype of infection in a potent and type-specific manner; however, most Abs generated in response to infection are serotype cross-reactive and poorly neutralizing. Such cross-reactive Abs may enhance disease during subsequent infection with a virus of a different DENV serotype. Previous screening assays for DENV-specific human B cells and antibodies, using viral and recombinant antigens, mainly led to the isolation of dominant nonneutralizing B cell clones. To improve upon our ability to recover and study rare but durable and potently neutralizing DENV-specific Abs, we isolated human DENV-specific B cells by using a primary screen of binding to live virus, followed by a secondary screen with a high-throughput, flow cytometry-based neutralization assay to identify DENV-specific B cell lines prior to generation of hybridomas. Using this strategy, we identified several new classes of serotype-specific and serotype-cross-neutralizing anti-DENV monoclonal Abs (MAbs), including ultrapotent inhibitory antibodies with neutralizing activity concentrations of IMPORTANCE Dengue virus infection is one of the most common mosquito-borne diseases and occurs in most countries of the world. Infection of humans with dengue virus induces a small number of antibodies that inhibit the infecting strain but also induces a large number of antibodies that can bind but do not inhibit dengue virus strains of other serotypes. We used a focused screening strategy to discover a large number of rare potently inhibiting antibodies, and we mapped the regions on the virus that were recognized by such antibodies. Our studies revealed that humans have the potential to generate very potent antibodies directed to diverse regions of the dengue virus surface protein. These studies provide important new information about protection from dengue virus infection that will be useful in the design and testing of new experimental dengue vaccines for humans.

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.

The immunopathogenesis of severe dengue is poorly understood, but there is concern that induction of cross-reactive nonneutralizing antibodies by infection or vaccination may increase the likelihood of severe disease during a subsequent infection. We generated a total of 63 new human monoclonal antibodies to compare the B-cell response of subjects who received the National Institutes of Health live attenuated dengue vaccine rDEN1Δ30 to that of subjects following symptomatic primary infection with DENV1. Both infection and vaccination induced serum neutralizing antibodies and DENV1-reactive peripheral blood B cells, but the magnitude of induction was lower in vaccinated individuals. Serotype cross-reactive weakly neutralizing antibodies dominated the response in both vaccinated and naturally infected subjects. Antigen specificities were very similar, with a slightly greater percentage of antibodies targeting E protein domain I/II than domain III. These data shed light on the similarity of human B-cell response to live attenuated DENV vaccine or natural infection.

The four dengue virus (DENV) serotypes, DENV1 through 4, are endemic throughout tropical and subtropical regions of the world. While first infection confers long-term protective immunity against viruses of the infecting serotype, a second infection with virus of a different serotype carries a greater risk of severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. Recent studies demonstrate that humans exposed to DENV infections develop neutralizing antibodies that bind to quaternary epitopes formed by the viral envelope (E) protein dimers or higher-order assemblies required for the formation of the icosahedral viral envelope. Here we show that the quaternary epitope target of the human DENV3-specific neutralizing monoclonal antibody (MAb) 5J7 can be partially transplanted into a DENV1 strain by changing the core residues of the epitope contained within a single monomeric E molecule. MAb 5J7 neutralized the recombinant DENV1/3 strain in cell culture and was protective in a mouse model of infection with the DENV1/3 strain. However, the 5J7 epitope was only partially recreated by transplantation of the core residues because MAb 5J7 bound and neutralized wild-type (WT) DENV3 better than the DENV1/3 recombinant. Our studies demonstrate that it is possible to transplant a large number of discontinuous residues between DENV serotypes and partially recreate a complex antibody epitope, while retaining virus viability. Further refinement of this approach may lead to new tools for measuring epitope-specific antibody responses and new vaccine platforms. IMPORTANCE Dengue virus is the most important mosquito-borne pathogen of humans worldwide, with approximately one-half the world's population living in regions where dengue is endemic. Dengue immunity following infection is robust and thought to be conferred by antibodies raised against the infecting virus. However, the specific viral components that these antibodies recognize and how they neutralize the virus have been incompletely described. Here we map a region on dengue virus serotype 3 recognized by the human neutralizing antibody 5J7 and then test the functional significance of this region by transplanting it into a serotype 1 virus. Our studies demonstrate a region on dengue virus necessary for 5J7 binding and neutralization. Our work also demonstrates the technical feasibility of engineering dengue viruses to display targets of protective antibodies. This technology can be used to develop new dengue vaccines and diagnostic assays.

The four dengue virus (DENV) serotypes, DENV-1, -2, -3, and -4, are endemic throughout tropical and subtropical regions of the world, with an estimated 390 million acute infections annually. Infection confers long-term protective immunity against the infecting serotype, but secondary infection with a different serotype carries a greater risk of potentially fatal severe dengue disease, including dengue hemorrhagic fever and dengue shock syndrome. The single most effective measure to control this threat to global health is a tetravalent DENV vaccine. To date, attempts to develop a protective vaccine have progressed slowly, partly because the targets of type-specific human neutralizing antibodies (NAbs), which are critical for long-term protection, remain poorly defined, impeding our understanding of natural immunity and hindering effective vaccine development. Here, we show that the envelope glycoprotein domain I/II hinge of DENV-3 and DENV-4 is the primary target of the long-term type-specific NAb response in humans. Transplantation of a DENV-4 hinge into a recombinant DENV-3 virus showed that the hinge determines the serotype-specific neutralizing potency of primary human and nonhuman primate DENV immune sera and that the hinge region both induces NAbs and is targeted by protective NAbs in rhesus macaques. These results suggest that the success of live dengue vaccines may depend on their ability to stimulate NAbs that target the envelope glycoprotein domain I/II hinge region. More broadly, this study shows that complex conformational antibody epitopes can be transplanted between live viruses, opening up similar possibilities for improving the breadth and specificity of vaccines for influenza, HIV, hepatitis C virus, and other clinically important viral pathogens.