Editorial
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Dengue virus envelope protein domain I/II hinge: a key target for dengue virus vaccine design? Expert Rev. Vaccines 14(1), 5–8 (2015)
Douglas G Widman Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA
Ralph S Baric Author for correspondence: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA [email protected]
Dengue virus is the most significant arboviral pathogen worldwide with nearly 400 million infections annually and half the global population at risk of disease. Despite this tremendous public health burden, there are no licensed treatments or vaccines to prevent dengue in humans. Results from clinical trials of leading vaccine candidates have demonstrated that our current understanding of the correlates of protection from dengue is incomplete, and as such vaccine performance has been moderate, but with considerable room for improvement. Here we highlight new findings revealing key neutralizing epitopes that regulate serotype-specific immunity, and discuss their implications for design and evaluation of future vaccine candidates. Challenges in the development of a tetravalent dengue vaccine
Despite over 40 years of research into the development of an efficacious tetravalent vaccine to prevent dengue virus (DENV), no vaccine formulations have achieved final approval for use in humans. At the root of this problem is the existence of four distinct serotypes of DENV (DENV1, 2, 3, 4) capable of sequential infections in humans, a characteristic unique among flaviviruses. While primary infection typically provides lifelong immunity to the infecting serotype, monotypic immunity to the original infecting serotype predisposes individuals to a higher risk of severe disease upon infection with heterologous serotypes. At the crux of this increased risk is the pathogenic nature of cross-reactive and nonneutralizing antibodies (Abs) elicited by primary DENV infection. These Abs facilitate infection of otherwise nonpermissive cells through a phenomenon known as antibody-dependent enhancement of infection, which has long been implicated as a factor in the development of severe DENV disease. As such, a safe and efficacious vaccine formulation must
provide strong immunity to all four serotypes simultaneously. Recently, the hopes of the DENV research field were raised by the entry of CYD, a liveattenuated DENV vaccine candidate that expresses the DENV prM and E proteins in the context of the attenuated yellow fever 17D (YFV17D) vaccine backbone, into Phase IIb [1] and Phase III [2,3] clinical trials. Unfortunately, results of the Phase IIb trial demonstrated a lack of protection against DENV2 disease, despite seroconversion rates and geometric mean neutralization titers at or exceeding those of other serotypes. Recent results from a Phase III trial showed slightly improved but still low levels of protective efficacy against symptomatic DENV2 disease. Perhaps the most troubling conclusions to be gleaned from these studies relate to the manner in which the correlates of protection for DENV are assessed by the field. These data clearly show that the traditional in vitro methods by which neutralizing Abs are measured lack significant correlation, perhaps in a serotypic specific manner, to in vivo protection from disease. A number of different conclusions could be drawn from these results. First, vaccination generated a population of
KEYWORDS: antibodies • dengue • epitope • protection • serotype • vaccine
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type-specific neutralizing Abs capable of blocking cell culturederived DENV infection in vitro; however, these Abs were incapable of providing protection from disease in vivo. This could be a result of differing maturation states of DENV grown in vitro versus that seen circulating in infected humans or differences in the mechanism of entry of target cells between the two systems, both of which have been hypothesized to play a role in the discrepancies observed between neutralization titers and correlates of protection from disease [4]. Second, it is possible that the CYD vaccine candidate did not generate DENV2-specific neutralizing Abs, but instead a population of cross-reactive Abs capable of neutralizing DENV2 in vitro, which were not protective in vivo. Fortunately, a 2-year follow-up showed no increase in severe disease in vaccinees, indicating that if the latter is indeed the case that this cross-reactive Ab population did not lead to disease enhancement. Additionally, levels of protection could rely on the magnitude of cell-mediated immune responses; however, these data are yet to be reported for these particular trials. Work with natural DENV infection has shown that CD4+ responses are directed primarily against moieties commonly targeted by B cells (C, E and NS1), while CD8+ responses recognize primarily NS3 and NS5 [5]. Due to the chimeric nature of CYD (the only DENV proteins it expresses are prM and E) the generation of DENV-specific T-cell responses may be difficult to achieve. Indeed, the results of a previous Phase II trial in Singapore demonstrated that while DENV-specific CD4+ and CD8+ responses could be detected, this was only in adult vaccinees who likely had a previous history of DENV infection, and thus these responses were probably the result of memory T-cell activation and not de novo induction of DENV-specific T-cell responses [6]. Furthermore, in the Phase IIb trial, CYD-induced viremia in YFV17Dimmune individuals was similar to that in naı¨ve subjects, calling into question the role of pre-existing CD4+ and CD8+ T-cell responses in controlling viral replication in human populations in the absence of neutralizing Abs [1]. Whichever the case, the results of this trial have revealed a current lack of knowledge of the correlates of protection from DENV disease and call for more fundamental and basic studies into the mechanisms and targets of human antibody neutralization. Such studies are critical as they will guide future investigations not only in vaccine design and administration, but the way in which we evaluate the protective efficacy of these candidates. One of the interesting results from the CYD clinical trials that may alter the way we fundamentally think about immunity to DENV infection relates to how humans respond to subsequent rounds of flavivirus infection. In an earlier Phase II trial of CYD, researchers found that prior immunity to flaviviruses, be it from natural DENV infection or from Japanese encephalitis or YFV17D vaccination, resulted in a more rapid and broad immune response to the DENV vaccine components than in naı¨ve individuals [7–9]. These results fit well with the dogma that secondary DENV infection elicits a population of strongly neutralizing cross-reactive Abs that provides protection from future infections. The breakthrough DENV2 infections observed in the Phase IIb results indicated that vaccination 6
with the tetravalent CYD vaccine in a population with previous flavivirus exposure history (>90% in this study) did not elicit a population of cross-reactive neutralizing Abs capable of protection as was previously believed, although the data were not parsed out to specifically compare responses in pre-immune and naı¨ve individuals. The Phase III trial did make this comparison, however, and found that the vaccine was far more efficacious in DENV seropositive individuals compared to naı¨ve subjects (74.3 vs 35.5%) [3], further suggesting that, as with T-cell responses, protection was largely mediated by a vaccineboosted pre-existing immune response rather than de novo vaccine-mediated immunity. These conflicting results indicate that further investigation is necessary, but suggest that immunity to heterologous flaviviruses may be required to augment immunity following DENV vaccination. Taken together, these results demonstrate that the development of a safe and efficacious tetravalent DENV vaccine for human use will require a much more detailed characterization of the complex Ab repertoire responsible for protection from disease after both primary and secondary infection. Human antibody responses to DENV infection
A number of factors have made detailed characterization of the antibody response to DENV infection difficult to ascertain, thus our understanding remains incomplete. Early studies using mice indicated that the majority of DENV-neutralizing Abs were targeted to E domain III (EDIII) (reviewed in [10]); however, recent studies indicated that this is not the case in human subjects. Depletion of human immune sera with recombinant EDIII had a minimal effect on the overall neutralization response to DENV, strongly indicating that Abs critical to serotype-specific immunity are directed to other regions of the E protein [11]. This also demonstrated that, while the field has long known that murine models are poor systems for studying DENV pathogenesis, the predominant immune responses appear to be fundamentally different than those mounted by humans, and as such, the translation of outcomes from murine to primate models must be viewed with caution. A further complication is that non-human primate (NHP) models replicate viruses about 1000-fold less efficiently than humans and do not produce appropriate disease outcomes, thus correlates of protection may well be easier to achieve in primates and differ in nature or magnitude with those correlates needed to achieve human protection. At the very least, these observations enforce the need for more detailed studies using human samples, including the development of human challenge models, before a more robust understanding of the correlates of protection to DENV disease are revealed to the vaccine community. Identification of DENV EDIII as a minor target of human neutralization responses was a significant finding that refocused interest toward other regions of the DENV virion to which strongly neutralizing type-specific Abs were elicited during human infection. A strong corollary to these results emerged when it was demonstrated that depletion of human immune sera with recombinant DENV (rDENV) E protein containing Expert Rev. Vaccines 14(1), (2015)
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Dengue virus envelope protein domain I/II hinge
Editorial
all three ectodomains had virtually no effect on the overall neutralization profile of these sera. These data indicated that critical neutralizing epitopes were primarily preserved only on intact virions and likely existed as complex quaternary epitopes spanning multiple E protein monomers [12]. We and others are focused on elucidating the human antibody repertoire, following primary and secondary infection to attempt to identify those epitopes that appeared critical for type-specific and broadly cross-reactive immunity.
provides greater insight into the human immune response to DENV infection. The results of these and future studies have the potential to inform the field regarding vaccine development and evaluation of potential vaccine candidates. We note that downstream studies evaluating particle maturation states on neutralization outcomes still remain to be performed with these recombinant viruses, thus it is possible that other strongly neutralizing sites exist within DENV that are not being revealed in this classic neutralization assay because of particle dynamics.
Identification of the EDI/II hinge region as a critical target for serotype-specific antibody neutralization
Considerations for vaccine development
Recent advancements in the generation and characterization of monoclonal antibodies (mAbs) from human donors have illuminated the diverse repertoire of Abs generated by natural DENV infection [13]. In particular, we and others have identified a number of strongly neutralizing serotype-specific mAbs that demonstrate complex patterns of binding. Specifically, most of these mAbs appear to bind epitopes present only on assembled virions [12]. Escape viruses generated by antibody selective pressure with these mAbs indicate that they localize to a complex quaternary epitope surrounding the DENV1 and DENV3 EDI/ II hinge region that undergoes complex structural rearrangements in the low pH environment of the late endosome to promote virus fusion and entry. Studies with a DENV3-specific mAb escape mutant showed a surprising decrease in neutralization sensitivity to human DENV3 polyclonal sera samples. Using residues identified in the escape mutants as a guide, and a recently developed infectious clone system for rDENV generation [14], a hypothetical conformational mAb epitope was identified using spatial dynamic analysis and the amino acid residues within this epitope were transplanted from DENV4 into DENV3 [15]. Significantly, this rDENV3/4 virus demonstrated increased sensitivity to neutralization by DENV4 immune sera from both naturally infected humans and experimentally infected NHP. Furthermore, the rDENV3/4 virus demonstrated reduced neutralization from a panel of DENV3 sera from humans and NHP. Studies have also identified the EDI/II hinge region of DENV1 as a critical component of the type-specific immune response elicited by a monovalent live-attenuated DENV1 vaccine candidate administered in a Phase I trial [16]. These results suggest the presence of a hierarchy of ‘master epitopes’ of serotype-specific immunity for DENV1 and DENV3 that primarily exist in the EDI/II hinge region, although it is less clear whether other serotypes encode similar sites in the E glycoprotein. The reduction of DENV3 neutralization sensitivity by a large panel of human and NHP sera and of DENV1 neutralization by sera from vaccinated humans indicates that this region is largely responsible for DENV1- and DENV3-specific neutralization following natural infection and/ or vaccination. DENV4 neutralization also maps to this region, although more recent studies suggest the presence of additional strong neutralizing domains elsewhere in the DENV4 virion as well. The identification of these regions/epitopes as a critical component of the human antibody response to DENV infection informahealthcare.com
As discussed above, a great deal of experimental data from human infections indicate that Ab-mediated serotype-specific neutralizing Ab responses following DENV infection are directed at complex quaternary epitopes present only on intact virions. If these observations are proven to correlate with in vivo protection from disease, they will have far-reaching effects on the design and implementation of future DENV vaccines. In this scenario, it seems unlikely that adjuvanted subunit vaccines or DENV antigens expressed from viral vectors or DNA vaccines would be capable of inducing neutralizing Ab responses directed at these highly conformational, virion-specific epitopes. In parallel, recent advances illuminating DENV particle breathing and E glycoprotein conformational plasticity [17] may further complicate evaluation of vaccine performance, especially when using structurally distinct virus-like particle [18] vaccines to mimic infectious DENV virions. E glycoprotein plasticity and particle maturation heterogeneity may further complicate detailed evaluation of the long-lived neutralizing responses associated with protective immunity even within the context of whole virion-based live-attenuated vaccines. Thus, despite significant advances in our understanding of the human antibody response to DENV infection and how these play a role in protection from disease, much fundamental work remains to be achieved before a highly efficacious tetravalent human vaccine is available for human populations. Future perspectives
The future of DENV vaccines relies on our ability to develop new techniques and diagnostic tools to measure and replicate human immunity to natural DENV infection. The most advanced current vaccine candidate recently demonstrated an overall protective efficacy of 56.5% against all four serotypes, with DENV2 efficacy falling far below this mark (35%) [3]. Furthermore, a recent Phase I trial of a tetravalent DENV vaccine candidate consisting of an attenuated DENV2 backbone containing prM/E genes of variant serotypes revealed similar problems. While demonstrating acceptable safety, neutralization titers to DENV2 predominated, while DENV4 titers were barely detectable under most inoculation conditions [19]. These results serve as a reminder that chimerization schemes must be carefully considered to preserve viral fitness necessary for induction of balanced immune responses against all four serotypes. As such, more work remains to be done to achieve more uniform protection from DENV 7
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Widman & Baric
disease. To this end, the identification of strongly neutralizing conformation-specific epitopes that serve as important determinants of serotype-specific neutralization is a critical advancement in the DENV field. With the recent development of a human challenge model for DENV vaccine evaluation [20], developing a library of diagnostic tools by which all vaccine candidates can be evaluated is critical. As demonstrated by previous studies, no longer can we rely on classical methods to make judgments on vaccine efficacy in complex human populations with varying degrees of prior immunity. Specific correlates of DENV immunity, such as Abs targeting the EDI/II hinge and other defined regions, must be identified and assays developed to assess their
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Sabchareon A, Wallace D, Sirivichayakul C, et al. Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 2012;380(9853):1559-67 Hss AS, Koh MT, Tan KK, et al. Safety and immunogenicity of a tetravalent dengue vaccine in healthy children aged 2-11 years in Malaysia: a randomized, placebo-controlled, Phase III study. Vaccine 2013;31(49):5814-21 Capeding MR, Tran NH, Hadinegoro SR, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014. [Epub ahead of print]
Financial & competing interests disclosure
The authors are inventors of pending patents related to this subject matter. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes. Hum Vaccin 2006;2(2): 60-7
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abundance in the sera of vaccinated individuals. This knowledge, as well as that relating to measures of protective efficacy, will someday facilitate the development of safe and effective DENV vaccines.
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Poo J, Galan F, Forrat R, et al. Live-attenuated Tetravalent Dengue Vaccine in Dengue-naive Children, Adolescents, and Adults in Mexico City: randomized Controlled Phase 1 Trial of Safety and Immunogenicity. Pediatr Infect Dis J 2010. [Epub ahead of print] Capeding RZ, Luna IA, Bomasang E, et al. Live-attenuated, tetravalent dengue vaccine in children, adolescents and adults in a dengue endemic country: randomized controlled phase I trial in the Philippines. Vaccine 2011;29(22):3863-72
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Dowd KA, Pierson TC. Antibody-mediated neutralization of flaviviruses: a reductionist view. Virology 2011;411(2):306-15
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Rivino L, Kumaran EA, Jovanovic V, et al. Differential targeting of viral components by CD4+ versus CD8+ T lymphocytes in dengue virus infection. J Virol 2013;87(5): 2693-706
Wahala WM, Kraus AA, Haymore LB, et al. Dengue virus neutralization by human immune sera: role of envelope protein domain III-reactive antibody. Virology 2009;392(1):103-13
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Harenberg A, Begue S, Mamessier A, et al. Persistence of Th1/Tc1 responses one year after tetravalent dengue vaccination in adults and adolescents in Singapore. Hum Vaccin Immunother 2013;9(11):2317-25
de Alwis R, Smith SA, Olivarez NP, et al. Identification of human neutralizing antibodies that bind to complex epitopes on dengue virions. Proc Natl Acad Sci USA 2012;109(19):7439-44
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Smith SA, de Alwis R, Kose N, et al. Human monoclonal antibodies derived from memory B cells following live attenuated dengue virus vaccination or natural infection exhibit similar characteristics. J Infect Dis 2013;207(12):1898-908
Mukherjee S, Dowd KA, Manhart CJ, et al. Mechanism and Significance of Cell Type-Dependent Neutralization of Flaviviruses. J Virol 2014;88(13):7210-20
Guirakhoo F, Kitchener S, Morrison D, et al. Live attenuated chimeric yellow fever dengue type 2 (ChimeriVax-DEN2) vaccine: phase I clinical trial for safety and immunogenicity: effect of yellow fever
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reverse genetic system for studying dengue virus serotype 3 strain variation and neutralization. PLoS Negl Trop Dis 2012; 6(2):e1486 15.
Messer WB, de Alwis R, Yount BL, et al. Dengue virus envelope protein domain I/II hinge determines long-lived serotype-specific dengue immunity. Proc Natl Acad Sci USA 2014;111(5):1939-44
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VanBlargan LA, Mukherjee S, Dowd KA, et al. The type-specific neutralizing antibody response elicited by a dengue vaccine candidate is focused on two amino acids of the envelope protein. PLoS Pathog 2013; 9(12):e1003761
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Lok SM, Kostyuchenko V, Nybakken GE, et al. Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat Struct Mol Biol 2008;15(3):312-17
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Ferlenghi I, Clarke M, Ruttan T, et al. Molecular organization of a recombinant subviral particle from tick-borne encephalitis virus. Mol Cell 2001;7(3):593-602
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Osorio JE, Velez ID, Thomson C, et al. Safety and immunogenicity of a recombinant live attenuated tetravalent dengue vaccine (DENVax) in flavivirus-naive healthy adults in Colombia: a randomised, placebo-controlled, phase 1 study. Lancet Infect Dis 2014; 14(9):830-8
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Cassetti MC, Thomas SJ. Dengue human infection model: introduction. J Infect Dis 2014;209(Suppl 2):S37-9
Messer WB, Yount B, Hacker KE, et al. Development and characterization of a
Expert Rev. Vaccines 14(1), (2015)
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Dengue virus envelope protein domain I/II hinge: a key target for dengue virus vaccine design? Expert Rev. Vaccines 14(1), 5–8 (2015)
Douglas G Widman Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA
Ralph S Baric Author for correspondence: Department of Epidemiology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 7435, USA [email protected]
Dengue virus is the most significant arboviral pathogen worldwide with nearly 400 million infections annually and half the global population at risk of disease. Despite this tremendous public health burden, there are no licensed treatments or vaccines to prevent dengue in humans. Results from clinical trials of leading vaccine candidates have demonstrated that our current understanding of the correlates of protection from dengue is incomplete, and as such vaccine performance has been moderate, but with considerable room for improvement. Here we highlight new findings revealing key neutralizing epitopes that regulate serotype-specific immunity, and discuss their implications for design and evaluation of future vaccine candidates. Challenges in the development of a tetravalent dengue vaccine
Despite over 40 years of research into the development of an efficacious tetravalent vaccine to prevent dengue virus (DENV), no vaccine formulations have achieved final approval for use in humans. At the root of this problem is the existence of four distinct serotypes of DENV (DENV1, 2, 3, 4) capable of sequential infections in humans, a characteristic unique among flaviviruses. While primary infection typically provides lifelong immunity to the infecting serotype, monotypic immunity to the original infecting serotype predisposes individuals to a higher risk of severe disease upon infection with heterologous serotypes. At the crux of this increased risk is the pathogenic nature of cross-reactive and nonneutralizing antibodies (Abs) elicited by primary DENV infection. These Abs facilitate infection of otherwise nonpermissive cells through a phenomenon known as antibody-dependent enhancement of infection, which has long been implicated as a factor in the development of severe DENV disease. As such, a safe and efficacious vaccine formulation must
provide strong immunity to all four serotypes simultaneously. Recently, the hopes of the DENV research field were raised by the entry of CYD, a liveattenuated DENV vaccine candidate that expresses the DENV prM and E proteins in the context of the attenuated yellow fever 17D (YFV17D) vaccine backbone, into Phase IIb [1] and Phase III [2,3] clinical trials. Unfortunately, results of the Phase IIb trial demonstrated a lack of protection against DENV2 disease, despite seroconversion rates and geometric mean neutralization titers at or exceeding those of other serotypes. Recent results from a Phase III trial showed slightly improved but still low levels of protective efficacy against symptomatic DENV2 disease. Perhaps the most troubling conclusions to be gleaned from these studies relate to the manner in which the correlates of protection for DENV are assessed by the field. These data clearly show that the traditional in vitro methods by which neutralizing Abs are measured lack significant correlation, perhaps in a serotypic specific manner, to in vivo protection from disease. A number of different conclusions could be drawn from these results. First, vaccination generated a population of
KEYWORDS: antibodies • dengue • epitope • protection • serotype • vaccine
informahealthcare.com
10.1586/14760584.2015.961431
Ó 2015 Informa UK Ltd
ISSN 1476-0584
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Editorial
Widman & Baric
type-specific neutralizing Abs capable of blocking cell culturederived DENV infection in vitro; however, these Abs were incapable of providing protection from disease in vivo. This could be a result of differing maturation states of DENV grown in vitro versus that seen circulating in infected humans or differences in the mechanism of entry of target cells between the two systems, both of which have been hypothesized to play a role in the discrepancies observed between neutralization titers and correlates of protection from disease [4]. Second, it is possible that the CYD vaccine candidate did not generate DENV2-specific neutralizing Abs, but instead a population of cross-reactive Abs capable of neutralizing DENV2 in vitro, which were not protective in vivo. Fortunately, a 2-year follow-up showed no increase in severe disease in vaccinees, indicating that if the latter is indeed the case that this cross-reactive Ab population did not lead to disease enhancement. Additionally, levels of protection could rely on the magnitude of cell-mediated immune responses; however, these data are yet to be reported for these particular trials. Work with natural DENV infection has shown that CD4+ responses are directed primarily against moieties commonly targeted by B cells (C, E and NS1), while CD8+ responses recognize primarily NS3 and NS5 [5]. Due to the chimeric nature of CYD (the only DENV proteins it expresses are prM and E) the generation of DENV-specific T-cell responses may be difficult to achieve. Indeed, the results of a previous Phase II trial in Singapore demonstrated that while DENV-specific CD4+ and CD8+ responses could be detected, this was only in adult vaccinees who likely had a previous history of DENV infection, and thus these responses were probably the result of memory T-cell activation and not de novo induction of DENV-specific T-cell responses [6]. Furthermore, in the Phase IIb trial, CYD-induced viremia in YFV17Dimmune individuals was similar to that in naı¨ve subjects, calling into question the role of pre-existing CD4+ and CD8+ T-cell responses in controlling viral replication in human populations in the absence of neutralizing Abs [1]. Whichever the case, the results of this trial have revealed a current lack of knowledge of the correlates of protection from DENV disease and call for more fundamental and basic studies into the mechanisms and targets of human antibody neutralization. Such studies are critical as they will guide future investigations not only in vaccine design and administration, but the way in which we evaluate the protective efficacy of these candidates. One of the interesting results from the CYD clinical trials that may alter the way we fundamentally think about immunity to DENV infection relates to how humans respond to subsequent rounds of flavivirus infection. In an earlier Phase II trial of CYD, researchers found that prior immunity to flaviviruses, be it from natural DENV infection or from Japanese encephalitis or YFV17D vaccination, resulted in a more rapid and broad immune response to the DENV vaccine components than in naı¨ve individuals [7–9]. These results fit well with the dogma that secondary DENV infection elicits a population of strongly neutralizing cross-reactive Abs that provides protection from future infections. The breakthrough DENV2 infections observed in the Phase IIb results indicated that vaccination 6
with the tetravalent CYD vaccine in a population with previous flavivirus exposure history (>90% in this study) did not elicit a population of cross-reactive neutralizing Abs capable of protection as was previously believed, although the data were not parsed out to specifically compare responses in pre-immune and naı¨ve individuals. The Phase III trial did make this comparison, however, and found that the vaccine was far more efficacious in DENV seropositive individuals compared to naı¨ve subjects (74.3 vs 35.5%) [3], further suggesting that, as with T-cell responses, protection was largely mediated by a vaccineboosted pre-existing immune response rather than de novo vaccine-mediated immunity. These conflicting results indicate that further investigation is necessary, but suggest that immunity to heterologous flaviviruses may be required to augment immunity following DENV vaccination. Taken together, these results demonstrate that the development of a safe and efficacious tetravalent DENV vaccine for human use will require a much more detailed characterization of the complex Ab repertoire responsible for protection from disease after both primary and secondary infection. Human antibody responses to DENV infection
A number of factors have made detailed characterization of the antibody response to DENV infection difficult to ascertain, thus our understanding remains incomplete. Early studies using mice indicated that the majority of DENV-neutralizing Abs were targeted to E domain III (EDIII) (reviewed in [10]); however, recent studies indicated that this is not the case in human subjects. Depletion of human immune sera with recombinant EDIII had a minimal effect on the overall neutralization response to DENV, strongly indicating that Abs critical to serotype-specific immunity are directed to other regions of the E protein [11]. This also demonstrated that, while the field has long known that murine models are poor systems for studying DENV pathogenesis, the predominant immune responses appear to be fundamentally different than those mounted by humans, and as such, the translation of outcomes from murine to primate models must be viewed with caution. A further complication is that non-human primate (NHP) models replicate viruses about 1000-fold less efficiently than humans and do not produce appropriate disease outcomes, thus correlates of protection may well be easier to achieve in primates and differ in nature or magnitude with those correlates needed to achieve human protection. At the very least, these observations enforce the need for more detailed studies using human samples, including the development of human challenge models, before a more robust understanding of the correlates of protection to DENV disease are revealed to the vaccine community. Identification of DENV EDIII as a minor target of human neutralization responses was a significant finding that refocused interest toward other regions of the DENV virion to which strongly neutralizing type-specific Abs were elicited during human infection. A strong corollary to these results emerged when it was demonstrated that depletion of human immune sera with recombinant DENV (rDENV) E protein containing Expert Rev. Vaccines 14(1), (2015)
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Dengue virus envelope protein domain I/II hinge
Editorial
all three ectodomains had virtually no effect on the overall neutralization profile of these sera. These data indicated that critical neutralizing epitopes were primarily preserved only on intact virions and likely existed as complex quaternary epitopes spanning multiple E protein monomers [12]. We and others are focused on elucidating the human antibody repertoire, following primary and secondary infection to attempt to identify those epitopes that appeared critical for type-specific and broadly cross-reactive immunity.
provides greater insight into the human immune response to DENV infection. The results of these and future studies have the potential to inform the field regarding vaccine development and evaluation of potential vaccine candidates. We note that downstream studies evaluating particle maturation states on neutralization outcomes still remain to be performed with these recombinant viruses, thus it is possible that other strongly neutralizing sites exist within DENV that are not being revealed in this classic neutralization assay because of particle dynamics.
Identification of the EDI/II hinge region as a critical target for serotype-specific antibody neutralization
Considerations for vaccine development
Recent advancements in the generation and characterization of monoclonal antibodies (mAbs) from human donors have illuminated the diverse repertoire of Abs generated by natural DENV infection [13]. In particular, we and others have identified a number of strongly neutralizing serotype-specific mAbs that demonstrate complex patterns of binding. Specifically, most of these mAbs appear to bind epitopes present only on assembled virions [12]. Escape viruses generated by antibody selective pressure with these mAbs indicate that they localize to a complex quaternary epitope surrounding the DENV1 and DENV3 EDI/ II hinge region that undergoes complex structural rearrangements in the low pH environment of the late endosome to promote virus fusion and entry. Studies with a DENV3-specific mAb escape mutant showed a surprising decrease in neutralization sensitivity to human DENV3 polyclonal sera samples. Using residues identified in the escape mutants as a guide, and a recently developed infectious clone system for rDENV generation [14], a hypothetical conformational mAb epitope was identified using spatial dynamic analysis and the amino acid residues within this epitope were transplanted from DENV4 into DENV3 [15]. Significantly, this rDENV3/4 virus demonstrated increased sensitivity to neutralization by DENV4 immune sera from both naturally infected humans and experimentally infected NHP. Furthermore, the rDENV3/4 virus demonstrated reduced neutralization from a panel of DENV3 sera from humans and NHP. Studies have also identified the EDI/II hinge region of DENV1 as a critical component of the type-specific immune response elicited by a monovalent live-attenuated DENV1 vaccine candidate administered in a Phase I trial [16]. These results suggest the presence of a hierarchy of ‘master epitopes’ of serotype-specific immunity for DENV1 and DENV3 that primarily exist in the EDI/II hinge region, although it is less clear whether other serotypes encode similar sites in the E glycoprotein. The reduction of DENV3 neutralization sensitivity by a large panel of human and NHP sera and of DENV1 neutralization by sera from vaccinated humans indicates that this region is largely responsible for DENV1- and DENV3-specific neutralization following natural infection and/ or vaccination. DENV4 neutralization also maps to this region, although more recent studies suggest the presence of additional strong neutralizing domains elsewhere in the DENV4 virion as well. The identification of these regions/epitopes as a critical component of the human antibody response to DENV infection informahealthcare.com
As discussed above, a great deal of experimental data from human infections indicate that Ab-mediated serotype-specific neutralizing Ab responses following DENV infection are directed at complex quaternary epitopes present only on intact virions. If these observations are proven to correlate with in vivo protection from disease, they will have far-reaching effects on the design and implementation of future DENV vaccines. In this scenario, it seems unlikely that adjuvanted subunit vaccines or DENV antigens expressed from viral vectors or DNA vaccines would be capable of inducing neutralizing Ab responses directed at these highly conformational, virion-specific epitopes. In parallel, recent advances illuminating DENV particle breathing and E glycoprotein conformational plasticity [17] may further complicate evaluation of vaccine performance, especially when using structurally distinct virus-like particle [18] vaccines to mimic infectious DENV virions. E glycoprotein plasticity and particle maturation heterogeneity may further complicate detailed evaluation of the long-lived neutralizing responses associated with protective immunity even within the context of whole virion-based live-attenuated vaccines. Thus, despite significant advances in our understanding of the human antibody response to DENV infection and how these play a role in protection from disease, much fundamental work remains to be achieved before a highly efficacious tetravalent human vaccine is available for human populations. Future perspectives
The future of DENV vaccines relies on our ability to develop new techniques and diagnostic tools to measure and replicate human immunity to natural DENV infection. The most advanced current vaccine candidate recently demonstrated an overall protective efficacy of 56.5% against all four serotypes, with DENV2 efficacy falling far below this mark (35%) [3]. Furthermore, a recent Phase I trial of a tetravalent DENV vaccine candidate consisting of an attenuated DENV2 backbone containing prM/E genes of variant serotypes revealed similar problems. While demonstrating acceptable safety, neutralization titers to DENV2 predominated, while DENV4 titers were barely detectable under most inoculation conditions [19]. These results serve as a reminder that chimerization schemes must be carefully considered to preserve viral fitness necessary for induction of balanced immune responses against all four serotypes. As such, more work remains to be done to achieve more uniform protection from DENV 7
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disease. To this end, the identification of strongly neutralizing conformation-specific epitopes that serve as important determinants of serotype-specific neutralization is a critical advancement in the DENV field. With the recent development of a human challenge model for DENV vaccine evaluation [20], developing a library of diagnostic tools by which all vaccine candidates can be evaluated is critical. As demonstrated by previous studies, no longer can we rely on classical methods to make judgments on vaccine efficacy in complex human populations with varying degrees of prior immunity. Specific correlates of DENV immunity, such as Abs targeting the EDI/II hinge and other defined regions, must be identified and assays developed to assess their
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Sabchareon A, Wallace D, Sirivichayakul C, et al. Protective efficacy of the recombinant, live-attenuated, CYD tetravalent dengue vaccine in Thai schoolchildren: a randomised, controlled phase 2b trial. Lancet 2012;380(9853):1559-67 Hss AS, Koh MT, Tan KK, et al. Safety and immunogenicity of a tetravalent dengue vaccine in healthy children aged 2-11 years in Malaysia: a randomized, placebo-controlled, Phase III study. Vaccine 2013;31(49):5814-21 Capeding MR, Tran NH, Hadinegoro SR, et al. Clinical efficacy and safety of a novel tetravalent dengue vaccine in healthy children in Asia: a phase 3, randomised, observer-masked, placebo-controlled trial. Lancet 2014. [Epub ahead of print]
Financial & competing interests disclosure
The authors are inventors of pending patents related to this subject matter. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.
pre-immunity in induction of cross neutralizing antibody responses to all 4 dengue serotypes. Hum Vaccin 2006;2(2): 60-7
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Expert Rev. Vaccines 14(1), (2015)
