Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site

ABSTRACT The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells

Mapping the Human Memory B Cell and Serum Neutralizing Antibody Responses to Dengue Virus Serotype 4 Infection and Vaccination

ABSTRACT The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses responsible for dengue fever and dengue hemorrhagic fever. People exposed to DENV develop antibodies (Abs) that strongly neutralize the serotype responsible for infection. Historically, infection with DENV serotype 4 (DENV4) has been less common and less studied than infections with the other three serotypes. However, DENV4 has been responsible for recent large and sustained epidemics in Asia and Latin America. The neutralizing antibody responses and the epitopes targeted against DENV4 have not been characterized in human infection. In this study, we mapped and characterized epitopes on DENV4 recognized by neutralizing antibodies in people previously exposed to DENV4 infections or to a live attenuated DENV4 vaccine. To study the fine specificity of DENV4 neutralizing human antibodies, B cells from two people exposed to DENV4 were immortalized and screened to identify DENV-specific clones. Two human monoclonal antibodies (MAbs) that neutralized DENV4 were isolated, and their epitopes were finely mapped using recombinant viruses and alanine scan mutation array techniques. Both antibodies bound to quaternary structure epitopes near the hinge region between envelope protein domain I (EDI) and EDII. In parallel, to characterize the serum neutralizing antibody responses, convalescence-phase serum samples from people previously exposed to primary DENV4 natural infections or a monovalent DENV4 vaccine were analyzed. Natural infection and vaccination also induced serum-neutralizing antibodies that targeted similar epitope domains at the EDI/II hinge region. These studies defined a target of neutralizing antigenic site on DENV4 targeted by human antibodies following natural infection or vaccination. IMPORTANCE The four serotypes of dengue virus are the causative agents of dengue fever and dengue hemorrhagic fever. People exposed to primary DENV infections develop long-term neutralizing antibody responses, but these principally recognize only the infecting serotype. An effective vaccine against dengue should elicit long-lasting protective antibody responses to all four serotypes simultaneously. We and others have defined antigenic sites on the envelope (E) protein of viruses of dengue virus serotypes 1, 2, and 3 targeted by human neutralizing antibodies. The epitopes on DENV4 E protein targeted by the human neutralizing antibodies and the mechanisms of serotype 4 neutralization are poorly understood. Here, we report the properties of human antibodies that neutralize dengue virus serotype 4. People exposed to serotype 4 infections or a live attenuated serotype 4 vaccine developed neutralizing antibodies that bound to similar sites on the viral E protein. These studies have provided a foundation for developing and evaluating DENV4 vaccines

Beyond Neutralizing Antibody Levels: The Epitope Specificity of Antibodies Induced by National Institutes of Health Monovalent Dengue Virus Vaccines

Background. Dengue virus is an emerging mosquito-borne flavivirus responsible for considerable morbidity and mortality worldwide. The Division of Intramural Research, National Institute of Allergy and Infectious Diseases of the US National Institutes of Health (NIH) has developed live attenuated vaccines to each of the 4 serotypes of dengue virus (DENV1–4). While overall levels of DENV neutralizing antibodies (nAbs) in humans have been correlated with protection, these correlations vary depending on DENV serotype, prevaccination immunostatus, age, and study site. By combining both the level and molecular specificity of nAbs to each serotype, it may be possible to develop more robust correlates that predict long-term outcome. Methods. Using depletions and recombinant chimeric epitope transplant DENVs, we evaluate the molecular specificity and mapped specific epitopes and antigenic regions targeted by vaccine-induced nAbs in volunteers who received the NIH monovalent vaccines against each DENV serotype. Results. After monovalent vaccination, subjects developed high levels of nAbs that mainly targeted epitopes that are unique (type-specific) to each DENV serotype. The DENV1, 2, and 4 monovalent vaccines induced type-specific nAbs directed to quaternary structure envelope epitopes known to be targets of strongly neutralizing antibodies induced by wild-type DENV infections. Conclusions. Our results reported here on the molecular specificity of NIH vaccine–induced antibodies enable new strategies, beyond the absolute levels of nAbs, for determining correlates and mechanisms of protective immunity

Genetic Variation between Dengue Virus Type 4 Strains Impacts Human Antibody Binding and Neutralization

There are four distinct DENV serotypes, and within DENV4, there are five distinct genotypes. The impact of genotypic diversity is not known, nor is it clear whether infection with one DENV4 genotype results in protective immunity against the other genotypes. To measure the impact of DENV4 genetic diversity, we generated an isogenic panel of viruses containing the envelope protein from the different genotypes. We characterized many properties of these viruses and find that a small number of amino acids changes within the envelope have disproportionate impacts on virus biology. Additionally, we observe large differences in the ability of DENV4 antibodies, immune sera, and vaccine sera to neutralize the panel, suggesting that DENV4 immunity might not be equally protective against all DENV4s. Our results support the monitoring of changing or emerging DENV genotypes and their role in escaping pre-existing neutralizing antibodies in people who have been vaccinated or exposed to natural DENV4 infections

Longitudinal analysis of acute and convalescent B cell responses in a human primary dengue serotype 2 infection model

Background: Acute viral infections induce a rapid and transient increase in antibody-secreting plasmablasts. At convalescence, memory B cells (MBC) and long-lived plasma cells (LLPC) are responsible for long-term humoral immunity. Following an acute viral infection, the specific properties and relationships between antibodies produced by these B cell compartments are poorly understood. Methods:Weutilized a controlled human challenge model of primary dengue virus serotype 2 (DENV2) infection to study acute and convalescent B-cell responses. Findings: The level of DENV2 replication was correlated with the magnitude of the plasmablast response. Functional analysis of plasmablast-derived monoclonal antibodies showed that the DENV2-specific response was dominated by cells producing DENV2 serotype-specific antibodies. DENV2-neutralizing antibodies targeted quaternary structure epitopes centered on domain III of the viral envelope protein (EDIII). Functional analysis ofMBC and serum antibodies from the same subjects six months post-challenge revealed maintenance of the serotypespecific response in both compartments. The serumresponse mainly targeted DENV2 serotype-specific epitopes on EDIII. Interpretation: Our data suggest overall functional alignment of DENV2-specific responses from the plasmablast, through the MBC and LLPC compartments following primary DENV2 inflection. These results provide enhanced resolution of the temporal and specificity of the B cell compartment in viral infection and serve as framework for evaluation of B cell responses in challenge models

High Rates of Hepatitis C Virus Reinfection and Spontaneous Clearance of Reinfection in People Who Inject Drugs: A Prospective Cohort Study

Hepatitis C virus reinfection and spontaneous clearance of reinfection were examined in a highly characterisedcohort of 188 people who inject drugs over a five-year period. Nine confirmed reinfections and 17 possiblereinfections were identified (confirmed reinfections were those genetically distinct from the previous infection andpossible reinfections were used to define instances where genetic differences between infections could not beassessed due to lack of availability of hepatitis C virus sequence data). The incidence of confirmed reinfection was28.8 per 100 person-years (PY), 95%CI: 15.0-55.4; the combined incidence of confirmed and possible reinfectionwas 24.6 per 100 PY (95%CI: 16.8-36.1). The hazard of hepatitis C reinfection was approximately double that ofprimary hepatitis C infection; it did not reach statistical significance in confirmed reinfections alone (hazard ratio [HR]:2.45, 95%CI: 0.87-6.86, p=0.089), but did in confirmed and possible hepatitis C reinfections combined (HR: 1.93,95%CI: 1.01-3.69, p=0.047) and after adjustment for the number of recent injecting partners and duration of injecting.In multivariable analysis, shorter duration of injection (HR: 0.91; 95%CI: 0.83-0.98; p=0.019) and multiple recentinjecting partners (HR: 3.12; 95%CI: 1.08-9.00, p=0.035) were independent predictors of possible and confirmedreinfection. Time to spontaneous clearance was shorter in confirmed reinfection (HR: 5.34, 95%CI: 1.67-17.03,p=0.005) and confirmed and possible reinfection (HR: 3.10, 95%CI: 1.10-8.76, p-value=0.033) than primary infection.Nonetheless, 50% of confirmed reinfections and 41% of confirmed or possible reinfections did not spontaneouslyclear.Conclusions: Hepatitis C reinfection and spontaneous clearance of hepatitis C reinfection were observed at highrates, suggesting partial acquired natural immunity to hepatitis C virus. Public health campaigns about the risks ofhepatitis C reinfection are required

T cell receptor repertoire recognition of hypervariable Hepatitis C virus

© 2012 Dr. Usha Kiranmayee NivarthiInfection with Hepatitis C virus (HCV) is a major public health problem, affecting more than 200 million people worldwide. 70% of the infected individuals progress to chronic infection and are at serious risk of developing liver cirrhosis and hepatocellular carcinoma. Nevertheless, all the components of immune response (innate, humoral and cellular) are shown to play a role in HCV immunity, the virus-specific CD4+ T cells and CD8+ T cells are thought to be the major anti-viral effector cells in HCV infection. Strong and vigorous T cell responses targeting multiple epitopes are associated with viral clearance. In chronic infection, HCV-specific CD8+ T cell responses are reported to be weak, narrowly focused and display functional defects in cytotoxicity, cytokine production and proliferative capacity. Different mechanisms that might contribute to the failure of HCV-specific CD8+ T cells in chronic infection have been identified. A clear understanding of the mechanisms contributing to T cell failure and persistence of HCV infection is important to the development of successful immunotherapeutic and prophylactic strategies. Mutations in CD8+ T- cell epitopes have been associated with immune escape and HCV persistence. Despite prolific mutations in CD8+T cell epitopes, there are very few studies on the T-cell receptor (TCR) repertoires targeted at these hypervariable HCV epitopes and none of them illustrate the structural interaction of the TCR and peptide-Human leukocyte antigen (pHLA) interactions corresponding to these hypervariable epitopes. Previous studies by our group demonstrated a narrow bias in the TCR repertoire targeted at the highly-mutated HLA-B*0801 restricted HSKKKCDEL (HSK), whilst a broad TCR repertoire was reported to be selected against comparatively-conserved HLA-A*0101 restricted ATDALMTGY (ATD), in HCV-infected, HLA-matched subjects with diverse disease-outcomes (natural HCV-clearance versus persistent-infection). To investigate if the narrow TCR repertoire selected against HSK contributed to CTL escape, a multidisciplinary study of T-cells targeted at the natural variants of HSK and its mutants reported in quasispecies isolated from chronically infected individuals was conducted. Functional analysis of T-cells was followed by surface plasmon resonance analysis of TCR-pHLA interactions and X-ray crystallography, aimed at solving the crystal structures of the binary complex and the ternary TCR-pHLA complex. The structural studies revealed for the first time the key HSK peptide residues that were in direct contact with the TCR and those that were buried in the groove of the HLA molecule, thus revealing the constraints or freedom the TCR has in accommodating the different natural variants. Functional data and SPR data validated the key residues that were critical for T-cell function revealing that any subtle change to residues at specific positions totally abrogated T-cell function. However, as mutations at these critical residues are reportedly rare and transient in nature, it is doubtful that escape through such rare mutations could cause HCV persistence. Further, the data demonstrated that despite a narrowly biased TCR repertoire, the TCRs used against HLA-B*0801/HSK not only facilitated cross-genotypic reactivity, but also permitted recognition of the common mutations in peptide determinants reported in the literature at different frequencies. Minor molecular changes to critical residues in viral epitopes usually impair normal T cell function. However, we still lack an understanding of the molecular basis of how conservative mutations at key residues affect the T cell function in human infection. In this regard, P6-C of the HSK peptide was found to be highly critical for T cell function. Further, our studies also revealed that P6-C is subject to biochemical modifications such as cysteinylation in RPMI and DMSO that could potentially decrease the antigenicity and immunogenicity of the peptide, and further affect the use of HSK, as a peptide based vaccine target. I sought to understand the effect of substitutions of cysteine in HSK on T cell recognition by replacing P6-C with more conservative amino acids like aminobutyric acid (Aba) and serine (isostereomers of cysteine). I also evaluated the potential of these synthetic peptide analogues as vaccine targets by studying their effect on T cell function. Structural and functional data demonstrated that HLA-B*0801/HSKKK(Aba)DEL binary complex had a comparable affinity for the specific TCR similar to the native peptide. However, cross-reactivity of T- cell lines directed towards the native peptide when tested on the modified aminobutyric acid was highly compromised despite only minor differences in binding and similar TRBV usage. Subtle differences in the ternary complex structures (differential orientation of the side groups of cysteine and aminobutyric acid) and minor differences in affinities had a drastic effect on T-cell functional abilities suggesting P6-C of HSK was important for optimal cellular responses and such peptide analogues must be used carefully for T cell based vaccines. Lastly, sequencing studies of the important NS3 protein coupled with functional data validated the previous observations that ATDALMTGY (ATD) is a relatively conserved epitope and that T-cells specific against the native ATD peptide were indeed cross-reactive to the natural variants, reported so far. Crystals of purified binary complex formed between the HLA molecule and the peptide (HLA-A*0101/ATD) were obtained. Further preliminary crystals formed by co crystallization of the purified TCR and the purified pHLA molecule, (ATD-TCR-HLA-HLA-A*0101/ATD) were obtained that may further reveal the interaction between the specific TCR and the ATD peptide at the atomic level. However, this aspect was beyond the scope of this doctoral study. Overall, the work described herein has provided a broader and enhanced understanding of the influence of TCR repertoire selection on the balance between CTL escape and host immunity in human HCV infection

Dengue Virus prM-Specific Human Monoclonal Antibodies with Virus Replication-Enhancing Properties Recognize a Single Immunodominant Antigenic Site

The proposed antibody-dependent enhancement (ADE) mechanism for severe dengue virus (DENV) disease suggests that non-neutralizing serotype cross-reactive antibodies generated during a primary infection facilitate entry into Fc receptor bearing cells during secondary infection, resulting in enhanced viral replication and severe disease. One group of cross-reactive antibodies that contributes considerably to this serum profile target the premembrane (prM) protein. We report here the isolation of a large panel of naturally occurring human monoclonal antibodies (MAbs) obtained from subjects following primary DENV serotype 1, 2, or 3 or secondary natural DENV infections or following primary DENV serotype 1 live attenuated virus vaccination to determine the antigenic landscape on the prM protein that is recognized by human antibodies. We isolated 25 prM-reactive human MAbs, encoded by diverse antibody-variable genes. Competition-binding studies revealed that all of the antibodies bound to a single major antigenic site on prM. Alanine scanning-based shotgun mutagenesis epitope mapping studies revealed diverse patterns of fine specificity of various clones, suggesting that different antibodies use varied binding poses to recognize several overlapping epitopes within the immunodominant site. Several of the antibodies interacted with epitopes on both prM and E protein residues. Despite the diverse genetic origins of the antibodies and differences in the fine specificity of their epitopes, each of these prM-reactive antibodies was capable of enhancing the DENV infection of Fc receptor-bearing cells. IMPORTANCE Antibodies may play a critical role in the pathogenesis of enhanced DENV infection and disease during secondary infections. A substantial proportion of enhancing antibodies generated in response to natural dengue infection are directed toward the prM protein. The fine specificity of human prM antibodies is not understood. Here, we isolated a panel of dengue prM-specific human monoclonal antibodies from individuals after infection in order to define the mode of molecular recognition by enhancing antibodies. We found that only a single antibody molecule can be bound to each prM protein at any given time. Distinct overlapping epitopes were mapped, but all of the epitopes lie within a single major antigenic site, suggesting that this antigenic domain forms an immunodominant region of the protein. Neutralization and antibody-dependent enhanced replication experiments showed that recognition of any of the epitopes within the major antigenic site on prM was sufficient to cause enhanced infection of target cells

Transplantation of a quaternary structure neutralizing antibody epitope from dengue virus serotype 3 into serotype 4

Abstract Dengue vaccine trials have revealed deficits in our understanding of the mechanisms of protective immunity, demonstrating a need to measure epitope-specific antibody responses against each DENV serotype. HmAb 5J7 binds to a complex, 3-monomer spanning quaternary epitope in the DENV3 envelope (E) protein, but it is unclear whether all interactions are needed for neutralization. Structure guided design and reverse genetics were used to sequentially transplant larger portions of the DENV3-specific 5J7 mAb epitope into dengue virus serotype 4 (DENV4). We observed complete binding and neutralization only when the entire 3 monomer spanning epitope was transplanted into DENV4, providing empirical proof that cooperative monomer-hmAb 5J7 interactions maximize activity. The rDENV4/3 virus containing the most expanded 5J7 epitope was also significantly more sensitive than WT DENV4 to neutralization by DENV3 primary immune sera. We conclude that the hinge-spanning region of the 5J7 quaternary epitope is a target for serotype-specific neutralizing antibodies after DENV3 infection

An extensive antigenic footprint underpins immunodominant TCR adaptability against a hypervariable viral determinant

Mutations in T cell epitopes are implicated in hepatitis C virus (HCV) persistence and can impinge on vaccine development. We recently demonstrated a narrow bias in the human TCR repertoire targeted at an immunodominant, but highly mutable, HLAB 0801-restricted epitope (HSKKKCDEL [HSK]). To investigate if the narrow TCR repertoire facilitates CTL escape, structural and biophysical studies were undertaken, alongside comprehensive functional analysis of T cells targeted at the natural variants of HLA-B∗0801-HSK in different HCV genotypes and quasispecies. Interestingly, within the TCR-HLA-B∗0801-HSK complex, the TCR contacts all available surface-exposed residues of the HSK determinant. This broad epitope coverage facilitates cross-genotypic reactivity and recognition of common mutations reported in HCV quasispecies, albeit to a varying degree. Certain mutations did abrogate T cell reactivity; however, natural variants comprising these mutations are reportedly rare and transient in nature, presumably due to fitness costs. Overall, despite a narrow bias, the TCR accommodated frequent mutations by acting like a blanket over the hypervariable epitope, thereby providing effective viral immunity. Our findings simultaneously advance the understanding of anti-HCV immunity and indicate the potential for cross-genotype HCV vaccines