Cell-Mediated Immunity Generated in Response to a Purified Inactivated Vaccine for Dengue Virus Type 1

Dengue is the most prevalent arboviral disease afflicting humans, and a vaccine appears to be the most rational means of control. Dengue vaccine development is in a critical phase, with the first vaccine licensed in some countries where dengue is endemic but demonstrating insufficient efficacy in immunologically naive populations. Since virus-neutralizing antibodies do not invariably correlate with vaccine efficacy, other markers that may predict protection, including cell-mediated immunity, are urgently needed. Previously, the Walter Reed Army Institute of Research developed a monovalent purified inactivated virus (PIV) vaccine candidate against dengue virus serotype 1 (DENV-1) adjuvanted with alum. The PIV vaccine was safe and immunogenic in a phase I dose escalation trial in healthy, flavivirus-naive adults in the United States. From that trial, peripheral blood mononuclear cells obtained at various time points pre- and postvaccination were used to measure DENV-1-specific T cell responses. After vaccination, a predominant CD4+ T cell-mediated response to peptide pools covering the DENV-1 structural proteins was observed. Over half (13/20) of the subjects produced interleukin-2 (IL-2) in response to DENV peptides, and the majority (17/20) demonstrated peptide-specific CD4+ T cell proliferation. In addition, analysis of postvaccination cell culture supernatants demonstrated an increased rate of production of cytokines, including gamma interferon (IFN-γ), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Overall, the vaccine was found to have elicited DENV-specific CD4+ T cell responses as measured by enzyme-linked immunosorbent spot (ELISpot), intracellular cytokine staining (ICS), lymphocyte proliferation, and cytokine production assays. Thus, together with antibody readouts, the use of a multifaceted measurement of cell-mediated immune responses after vaccination is a useful strategy for more comprehensively characterizing immunity generated by dengue vaccines

Vaccine Protection Against Zika Virus from Brazil

Zika virus (ZIKV) is a flavivirus that is responsible for an unprecedented current epidemic in Brazil and the Americas1,2. ZIKV has been causally associated with fetal microcephaly, intrauterine growth restriction, and other birth defects in both humans3–8 and mice9–11. The rapid development of a safe and effective ZIKV vaccine is a global health priority1,2, but very little is currently known about ZIKV immunology and mechanisms of immune protection. Here we show that a single immunization of a plasmid DNA vaccine or a purified inactivated virus vaccine provides complete protection in susceptible mice against challenge with a ZIKV outbreak strain from northeast Brazil. This ZIKV strain has recently been shown to cross the placenta and to induce fetal microcephaly and other congenital malformations in mice11. We produced DNA vaccines expressing full-length ZIKV pre-membrane and envelope (prM-Env) as well as a series of deletion mutants. The full-length prM-Env DNA vaccine, but not the deletion mutants, afforded complete protection against ZIKV as measured by absence of detectable viremia following challenge, and protective efficacy correlated with Env-specific antibody titers. Adoptive transfer of purified IgG from vaccinated mice conferred passive protection, and CD4 and CD8 T lymphocyte depletion in vaccinated mice did not abrogate protective efficacy. These data demonstrate that protection against ZIKV challenge can be achieved by single-shot subunit and inactivated virus vaccines in mice and that Env-specific antibody titers represent key immunologic correlates of protection. Our findings suggest that the development of a ZIKV vaccine for humans will likely be readily achievable

Immunological Assays used to Support Efficacy of Zika Virus Vaccines

In February of 2016, the World Health Organization (WHO) declared Zika virus (ZIKV) a Public Health Emergency of International Concern. This prompted a rapid response from both the private and public sector resulting in the generation of several promising vaccine candidates. In this review, we discuss published scientific efforts associated with these novel vaccines, emphasizing the immunological assays used to evaluate their immunogenicity and efficacy, and support future licensure

Passage of Dengue Virus Type 4 Vaccine Candidates in Fetal Rhesus Lung Cells Selects Heparin-Sensitive Variants That Result in Loss of Infectivity and Immunogenicity in Rhesus Macaques▿

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3′ noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu327-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu327-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu327-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu327-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy

Cell-mediated immune responses to different formulations of a live-attenuated tetravalent dengue vaccine candidate in subjects living in dengue endemic and non-endemic regions

Three phase II randomized trials evaluated the safety/immunogenicity of two formulations of live-attenuated tetravalent dengue virus (TDEN) vaccine in dengue-endemic (Puerto Rico, Thailand) and non-endemic (US) regions (NCT00350337/NCT00370682/NCT00468858). We describe cell-mediated immune (CMI) responses; safety and humoral responses were reported previously. Participants received two doses of vaccine or control (placebo or the precursor live-attenuated TDEN vaccine) 6 months apart. Selected US participants received a booster 5–12 months post-dose 2. Evaluated subsets of the per-protocol cohorts included 75 primarily dengue virus (DENV)-unprimed US adults, 69 primarily flavivirus-primed Thai adults, and 100 DENV-primed or DENV-unprimed Puerto Rican adults/adolescents/children. T-cell responses were quantified using intracellular cytokine staining (ICS; DENV-infected cell-lysate or DENV-1/DENV-2 peptide-pool stimulation) or IFN-γ ELISPOT (DENV-2 peptide-pool stimulation). Memory B-cell responses were quantified using B-cell ELISPOT. Across populations and age strata, DENV serotype-specific CD4+ T-cell responses were slightly to moderately increased (medians ≤0.18% [ICS]), DENV-2–biased, and variable for both formulations. Responses in unprimed subjects were primarily detected post-dose 1. Response magnitudes in primed subjects were similar between doses. Multifunctional CD8+ T-cell responses were detected after peptide-pool stimulation. T-cell responses were mostly directed to DENV nonstructural proteins 3 and 5. Memory B-cell responses were tetravalent, of low-to-moderate magnitudes (medians ≤0.25%), and mainly observed post-dose 2 in unprimed subjects and post-dose 1 in primed subjects. A third dose did not boost CMI responses. In conclusion, both formulations of the live-attenuated TDEN vaccine candidate were poorly to moderately immunogenic with respect to B-cell and T-cell responses, irrespective of the priming status of the participants. Abbreviation ATP: according-to-protocol; ICS: Intracellular Cytokine Staining; NS3: Nonstructural protein 3; ELISPOT: Enzyme-Linked ImmunoSpot; JEV: Japanese encephalitis virus; PBMC: peripheral blood mononuclear cell