Viral genetic diversity and protective efficacy of a tetravalent dengue vaccine in two phase 3 trials.

Two phase 3 placebo-controlled trials of the CYD-TDV vaccine, evaluated in children aged 2-14 y (CYD14) and 9-16 y (CYD15), demonstrated vaccine efficacy (VE) of 56.5% and 60.8%, respectively, against symptomatic virologically confirmed dengue (VCD). Sieve analyses were conducted to evaluate whether and how VE varied with amino acid sequence features of dengue viruses (DENVs). DENV premembrane/envelope amino acid sequences from VCD endpoint cases were aligned with the vaccine insert sequences, and extensions of the proportional hazards model were applied to assess variation in VE with amino acid mismatch proportion distances from vaccine strains, individual amino acid residues, and phylogenetic genotypes. In CYD14, VE against VCD of any serotype (DENV-Any) decreased significantly with increasing amino acid distance from the vaccine, whereas in CYD15, VE against DENV-Any was distance-invariant. Restricting to the common age range and amino acid distance range between the trials and accounting for differential VE by serotype, however, showed no evidence of VE variation with distance in either trial. In serotype-specific analyses, VE against DENV4 decreased significantly with increasing amino acid distance from the DENV4 vaccine insert and was significantly greater against residue-matched DENV4 at eight signature positions. These effects were restricted to 2- to 8-y-olds, potentially because greater seropositivity of older children at baseline might facilitate a broader protective immune response. The relevance of an antigenic match between vaccine strains and circulating DENVs was also supported by greater estimated VE against serotypes and genotypes for which the circulating DENVs had shorter amino acid sequence distances from the vaccine.

Vaccination Against Dengue: Challenges and Current Developments.

Dengue is a growing threat worldwide, and the development of a vaccine is a public health priority. The completion of the active phase of two pivotal efficacy studies conducted in Asia and Latin America by Sanofi Pasteur has constituted an important step. Several other approaches are under development, and whichever technology is used, vaccine developers face several challenges linked to the particular nature and etiology of dengue disease. We start our review by defining questions and potential issues linked to dengue pathology and presenting the main types of vaccine approaches that have explored these questions; some of these candidates are in a late stage of clinical development. In the second part of the review, we focus on the Sanofi Pasteur dengue vaccine candidate, describing the steps from research to phase III efficacy studies. Finally, we discuss what could be the next steps, before and after vaccine introduction, to ensure that the vaccine will provide the best benefit with an acceptable safety profile to the identified target populations.

Dissecting Antibodies Induced by a Chimeric Yellow Fever-Dengue, Live-Attenuated, Tetravalent Dengue Vaccine (CYD-TDV) in Naive and Dengue-Exposed Individuals.

Sanofi Pasteur has developed a chimeric yellow fever-dengue, live-attenuated, tetravalent dengue vaccine (CYD-TDV) that is currently approved for use in several countries. In clinical trials, CYD-TDV was efficacious at reducing laboratory-confirmed cases of dengue disease. Efficacy varied by dengue virus (DENV) serotype and prevaccination dengue immune status. We compared the properties of antibodies in naive and DENV-exposed individuals who received CYD-TDV. We depleted specific populations of DENV-reactive antibodies from immune serum samples to estimate the contribution of serotype-cross-reactive and type-specific antibodies to neutralization. Subjects with no preexisting immunity to DENV developed neutralizing antibodies to all 4 serotypes of DENV. Further analysis demonstrated that DENV4 was mainly neutralized by type-specific antibodies whereas DENV1, DENV2, and DENV3 were mainly neutralized by serotype cross-reactive antibodies. When subjects with preexisting immunity to DENV were vaccinated, they developed higher levels of neutralizing antibodies than naive subjects who were vaccinated. In preimmune subjects, CYD-TDV boosted cross-reactive neutralizing antibodies while maintaining type-specific neutralizing antibodies acquired before vaccination. Our results demonstrate that the quality of neutralizing antibodies induced by CYD-TDV varies depending on DENV serotype and previous immune status. We discuss the implications of these results for understanding vaccine efficacy.

Tetravalent Dengue Vaccine Reduces Symptomatic and Asymptomatic Dengue Virus Infections in Healthy Children and Adolescents Aged 2-16 Years in Asia and Latin America.

BACKGROUND: Asymptomatic dengue virus-infected individuals are thought to play a major role in dengue virus transmission. The efficacy of the recently approved quadrivalent CYD-TDV dengue vaccine against asymptomatic dengue virus infection has not been previously assessed. METHODS: We pooled data for 3 736 individuals who received either CYD-TDV or placebo at 0, 6, and 12 months in the immunogenicity subsets of 2 phase 3 trials (clinical trials registration NCT01373281 and NCT01374516). We defined a seroconversion algorithm (ie, a ≥4-fold increase in the neutralizing antibody titer and a titer of ≥40 from month 13 to month 25) as a surrogate marker of asymptomatic infection in the vaccine and placebo groups. RESULTS: The algorithm detected seroconversion in 94% of individuals with a diagnosis of virologically confirmed dengue between months 13 and 25, validating its discriminatory power. Among those without virologically confirmed dengue (n = 3 669), 219 of 2 485 in the vaccine group and 157 of 1 184 in the placebo group seroconverted between months 13 and 25, giving a vaccine efficacy of 33.5% (95% confidence interval [CI], 17.9%-46.1%) against asymptomatic infection. Vaccine efficacy was marginally higher in subjects aged 9-16 years (38.6%; 95% CI, 22.1%-51.5%). The annual incidence of asymptomatic dengue virus infection in this age group was 14.8%, which was 4.4 times higher than the incidence for symptomatic dengue (3.4%). CONCLUSIONS: The observed vaccine efficacy against asymptomatic dengue virus infections is expected to translate into reduced dengue virus transmission if sufficient individuals are vaccinated in dengue-endemic areas.

Cross-serotype immunity induced by immunization with a conserved rhinovirus capsid protein.

Human rhinovirus (RV) infections are the principle cause of common colds and precipitate asthma and COPD exacerbations. There is currently no RV vaccine, largely due to the existence of ∼150 strains. We aimed to define highly conserved areas of the RV proteome and test their usefulness as candidate antigens for a broadly cross-reactive vaccine, using a mouse infection model. Regions of the VP0 (VP4+VP2) capsid protein were identified as having high homology across RVs. Immunization with a recombinant VP0 combined with a Th1 promoting adjuvant induced systemic, antigen specific, cross-serotype, cellular and humoral immune responses. Similar cross-reactive responses were observed in the lungs of immunized mice after infection with heterologous RV strains. Immunization enhanced the generation of heterosubtypic neutralizing antibodies and lung memory T cells, and caused more rapid virus clearance. Conserved domains of the RV capsid therefore induce cross-reactive immune responses and represent candidates for a subunit RV vaccine.

Different innate signatures induced in human monocyte-derived dendritic cells by wild-type dengue 3 virus, attenuated but reactogenic dengue 3 vaccine virus, or attenuated nonreactogenic dengue 1-4 vaccine virus strains.

DNA microarrays were used to assess the innate gene signature in human myeloid dendritic cells infected with chimeric dengue 1-4 vaccines, a wild-type dengue 3 virus, or a classically attenuated serotype 3 vaccine shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 chimeric dengue vaccines, involving stimulation of type I interferon and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, wild-typeDEN3 virus induced a predominantly inflammatory profile, while the reactogenic attenuated serotype 3 vaccine appeared to induce a blunted response.

[Tetravalent dengue vaccine development]. / Développement d'un vaccin tétravalent contre la dengue.

The Sanofi Pasteur tetravalent dengue vaccine candidate (TV) is composed of four recombinant live attenuated vaccines based on a yellow fever virus vaccine 17D (YFV 17D) backbone, each expressing the prM and envelope genes of one of the four dengue virus serotypes. Pre-clinical studies have demonstrated that the TV dengue vaccine is genetically and phenotypi- cally stable, non-hepatotropic, less neurovirulent than YFV 17D and does not infect mosquitoes by the oral route. In clinics, TV dengue vaccine reactogeni- city, viraemia induction and antibody responses were investigated in three Phase II trials in endemic and non-endemic countries, which demonstrated its good safety and immunogenicity. An extensive clinical development program for dengue TV is underway including an efficacy trial in Thailand, in an area of high dengue incidence. Assuming continued successful outcomes, initial submissions to regulatory authorities are envisaged within a 5-year period.

When Can One Vaccinate with a Live Vaccine after Wild-Type Dengue Infection?

Recommendations have been issued for vaccinating with the Sanofi Pasteur tetravalent dengue vaccine (CYD-TDV, Dengvaxia®) individuals aged from 9 to 45/60 years old with a prior dengue virus (DENV) infection and living in endemic countries/areas. One question linked to these recommendations is to determine when it is possible to start vaccination after laboratory confirmed wild-type DENV infection, and this question can be relevant to any live vaccine to be used in endemic areas. To address it, we reviewed and discussed the immunological and practical considerations of live vaccination in this context. Firstly, the nature and kinetics of immune responses triggered by primary or secondary DENV infection may positively or negatively impact subsequent live vaccine take and associated clinical benefit, depending on when vaccination is performed after infection. Secondly, regarding practical aspects, the "easiest" situation would correspond to a confirmed acute dengue fever, only requiring knowing when the patient should come back for vaccination. However, in most cases, it will not be possible to firmly establish the actual date of infection and vaccination may have to take place during well-defined periods, regardless of when prior infection occurred. Evidence that informs health authorities and medical practitioners in formulating vaccine policies and implementing vaccine programs is thus needed. The present work reviewed the different elements of the guidance and proposes some key conclusions and recommendations.

Evaluation of rapid diagnostic tests and conventional enzyme-linked immunosorbent assays to determine prior dengue infection.

BACKGROUND: In September 2018, the World Health Organization recommended that prevaccination screening be used with the tetravalent dengue vaccine (CYD-TDV), to ensure that only individuals with evidence of prior dengue infection (PDI) are vaccinated. Dengue rapid diagnostic tests (RDTs) would offer a potential solution for prevaccination screening at the point-of-care, but data on performance of available RDTs for identifying PDI are limited. We determined the suitability of four dengue RDTs and two conventional enzyme-linked immunosorbent assays (ELISAs) to identify PDI and evaluated cross-reactivity with co-circulating flaviviruses.Methods: Specificity was assessed using 534 dengue-negative [determined by 50% plaque reduction neutralization test (PRNT50)] serum samples from USA (n = 229) and dengue-endemic regions (n = 305). Sensitivity was assessed using 270 samples from recent (n = 90) or remote (n = 90) virologically confirmed prior dengue cases, and dengue PRNT50-positive samples (n = 90). Cross-reactivity was assessed in dengue-seronegative samples that were seropositive for yellow fever (n = 57), Japanese encephalitis (n = 37), West Nile (n = 59) or Zika (n = 41).Results: Dengue IgG RDTs and the Panbio ELISA exhibited favourable specificities (99-100%), higher than the Focus ELISA (95%). The RDTs had variable sensitivities (40-70%) that were lower than those of the ELISAs (≥90%). Cross-reactivity to other flaviviruses was low with RDTs (≤7%), but more significant with ELISAs (up to 51% for West Nile and 34% for Zika). No cross-reactivity to any of the four closely related flaviviruses was observed with the CTK Biotech RDT. For each SeroTest, sensitivity appeared similar in samples from individuals with recent (<13 months) vs remote (3-4 years) virologically confirmed PDI.Conclusions: In general, dengue IgG RDTs were found to be more specific and less cross-reactive than the ELISAs, but the latter were more sensitive for identifying PDI cases. Currently available RDTs could be temporizing tools for rapid and safe prevaccination screening until improved RDTs with increased sensitivity become available.

Characterization of recombinant yellow fever-dengue vaccine viruses with human monoclonal antibodies targeting key conformational epitopes.

The recombinant yellow fever-17D-dengue virus, live, attenuated, tetravalent dengue vaccine (CYD-TDV) is licensed in several dengue-endemic countries. Although the vaccine provides protection against dengue, the level of protection differs by serotype and warrants further investigation. We characterized the antigenic properties of each vaccine virus serotype using highly neutralizing human monoclonal antibodies (hmAbs) that bind quaternary structure-dependent epitopes. Specifically, we monitored the binding of dengue virus-1 (DENV-1; 1F4), DENV-2 (2D22) or DENV-3 (5J7) serotype-specific or DENV-1-4 cross-reactive (1C19) hmAbs to the four chimeric yellow fever-dengue vaccine viruses (CYD-1-4) included in phase III vaccine formulations using a range of biochemical and functional assays (dot blot, ELISA, surface plasmon resonance and plaque reduction neutralization assays). In addition, we used the "classic" live, attenuated DENV-2 vaccine serotype, immature CYD-2 viruses and DENV-2 virus-like particles as control antigens for anti-serotype-2 reactivity. The CYD vaccine serotypes were recognized by each hmAbs with the expected specificity, moreover, surface plasmon resonance indicated a high functional affinity interaction with the CYD serotypes. In addition, the hmAbs provided similar protection against CYD and wild-type dengue viruses in the in vitro neutralization assay. Overall, these findings demonstrate that the four CYD viruses used in clinical trials display key conformational and functional epitopes targeted by serotype-specific and/or cross-reactive neutralizing human antibodies. More specifically, we showed that CYD-2 displays serotype- specific epitopes present only on the mature virus. This indicates that the CYD-TDV has the ability to elicit antibody specificities which are similar to those induced by the wild type DENV. Future investigations will be needed to address the nature of CYD-TDV-induced responses after vaccine administration, and how these laboratory markers relate to vaccine efficacy and safety.

Immunogenicity and safety of a quadrivalent plant-derived virus like particle influenza vaccine candidate-Two randomized Phase II clinical trials in 18 to 49 and ≥50 years old adults.

BACKGROUND: New influenza vaccines eliciting more effective protection are needed, particularly for the elderly who paid a large and disproportional toll of hospitalization and dead during seasonal influenza epidemics. Low (≤15 µg/strain) doses of a new plant-derived virus-like-particle (VLP) vaccine candidate have been shown to induce humoral and cellular responses against both homologous and heterologous strains in healthy adults 18-64 years of age. The two clinical trials reported here addressed the safety and immunogenicity of higher doses (≥15 µg/strain) of quadrivalent VLP candidate vaccine on 18-49 years old and ≥50 years old subjects. We also investigated the impact of alum on the immunogenicity induced by lower doses of the vaccine candidate. METHOD: In the first Phase II trial reported here (NCT02233816), 18-49 year old subjects received 15, 30 or 60 µg/strain of a hemagglutinin-bearing quadrivalent virus-like particle (QVLP) vaccine or placebo. In the second trial (NCT02236052), ≥50 years old subjects received QVLP as above or placebo with additional groups receiving 7.5 or 15 µg/strain with alum. Along with safety recording, the humoral and cell-mediated immune responses were analyzed. RESULTS: Local and systemic side-effects were similar to those reported previously. The QVLP vaccine induced significant homologous and heterologous antibody responses at the two higher doses, the addition of alum having little-to-no effect. Serologic outcomes tended to be lower in ≥50 years old subjects previously vaccinated. The candidate vaccine also consistently elicited both homologous and heterologous antigen-specific CD4+ T cells characterized by their production of interferon-gamma (IFN-γ), interleukine-2 (IL-2) and/or tumor-necrosis factor alpha (TNF-α) upon ex vivo antigenic restimulation. CONCLUSION: Overall, the 30 µg dose produced the most consistent humoral and cellular responses in both 18-49 and ≥50 years old subjects, strongly supporting the clinical development of this candidate vaccine. That particular dose was chosen to test in the ongoing Phase III clinical trial.

Complementary use of mass spectrometry and cryo-electron microscopy to assess the maturity of live attenuated dengue vaccine viruses.

Dengue virus (DENV) infection is a global health threat with the potential to affect at least 3.6 billion people living in areas of risk. No specific curative treatments against dengue disease are available and vaccines are currently the only way to prevent the disease. The tetravalent dengue vaccine developed by Sanofi Pasteur has demonstrated significant efficacy in phase III studies and is now licensed in several countries for the prevention of disease in dengue-seropositives over 9 years of age. The vaccine is composed of four recombinant, live, attenuated vaccines (CYD 1-4) based on a yellow fever vaccine 17D (YFV 17D) backbone, each expressing the pre-membrane (prM) and envelope (E) genes of one of the four DENV serotypes. Virus maturity could impact the biological activity of the vaccine viruses. To address this question, the maturity of the four vaccine viruses used in phase III clinical studies was assessed by two complementary techniques: mass spectrometry (MS) and cryo-electron microscopy (cryoEM). MS assessed viral maturity at the molecular level by quantifying specifically the prM, and M proteins. CryoEM provided information at the particle level, allowing visualizing the different phenotypes of viral particles: spiky (immature), smooth/bumpy (mature), and mixed (partially mature). Results of the two assays used in this study show that all four CYD dengue vaccine viruses present in lots used in phase III efficacy trials, display in the majority a mature phenotype.

Towards a dengue vaccine: progress to date and remaining challenges.

The increased incidence and extended geographical reach of Dengue virus over the past two decades have made the development of an effective vaccine an international urgency. Various strategies are being pursued, including live, vectored and killed/recombinant preparations. For all approaches, the challenge is to induce a broad durable immune response against all four serotypes of Dengue virus simultaneously whilst avoiding the possible exacerbation of risk of developing the severe forms of disease through incomplete or modified responses. This review presents the current state of knowledge and discusses the challenges of further clinical development.

Which Dengue Vaccine Approach Is the Most Promising, and Should We Be Concerned about Enhanced Disease after Vaccination? Questions Raised by the Development and Implementation of Dengue Vaccines: Example of the Sanofi Pasteur Tetravalent Dengue Vaccine.

Dengue is a still-growing public health concern in many tropical and subtropical regions of the world. The development and implementation of an effective dengue vaccine in these regions is a high priority. This insight focuses on the expected characteristics of a safe and efficacious vaccine, referring to the clinical experience obtained during the development of the first tetravalent dengue vaccine from Sanofi Pasteur, now licensed in several endemic countries. Safety and efficacy data from both short- and long-term follow-up of large-scale efficacy studies will be discussed, as well as the next steps following vaccine introduction.

Dengue seroprevalence: data from the clinical development of a tetravalent dengue vaccine in 14 countries (2005-2014).

Dengue seroprevalence data in the literature is limited and the available information is difficult to compare between studies because of the varying survey designs and methods used. We assessed dengue seropositivity across 14 countries using data from 15 trials conducted during the development of a tetravalent dengue vaccine between October 2005 and February 2014. Participants' dengue seropositivity (n=8592) was determined from baseline (before vaccination) serum samples at two centralized laboratories with the plaque reduction neutralization test (PRNT50). Seropositivity rates generally increased with age in endemic settings. Although seropositivity rates varied across geographical areas, between countries, and within countries by region, no major differences were observed for given age groups between the two endemic regions, Latin America and Asia-Pacific. Seropositivity rates were generally stable over time. The proportion of participants who had only experienced primary infection tended to be higher in younger children than adolescents/adults. These results will help inform and guide dengue control strategies in the participating countries.

Innate immune responses in human dendritic cells upon infection by chimeric yellow-fever dengue vaccine serotypes 1-4.

Dengue infection is an important public health issue worldwide. The ChimeriVax-Dengue (CYD) vaccine uses yellow fever (YF) 17D vaccine as a live vector. Dendritic cells (DCs) play a key role in initiating immune responses and could be an important primary target of dengue infection. We investigated in vitro the consequences of CYD infection of DCs on their activation/maturation and cytokine production. In CYD-infected DCs, we observed an up-regulation of HLA-DR, CD80, CD86, and CD83. Cells exposed to CYD secreted type I interferons, monocyte chemoattractant protein 1 (MCP-1)/CC chemokine ligand 2 (CCL-2), interleukin-6 (IL-6), and low amounts of tumor necrosis factor-alpha (TNF-alpha), but no IL-10, IL-12, or IL-1alpha. Parental dengue viruses induced a similar array of cytokines, but more TNF-alpha, less IL-6, and less MCP-1/CCL-2 than induced by CYD. Chimeras thus induced DCs maturation and a controlled response accompanied by limited inflammatory cytokine production and consistent expression of anti-viral interferons, in agreement with clinical observations of safety and immunogenicity.

Biodistribution and safety of a live attenuated tetravalent dengue vaccine in the cynomolgus monkey.

BACKGROUND: The first licensed dengue vaccine is a recombinant, live, attenuated, tetravalent dengue virus vaccine (CYD-TDV; Sanofi Pasteur). This study assessed the biodistribution, shedding, and toxicity of CYD-TDV in a non-human primate model as part of the nonclinical safety assessment program for the vaccine. METHODS: Cynomolgus monkeys were given one subcutaneous injection of either one human dose (5log10 CCID50/serotype) of CYD-TDV or saline control. Study endpoints included clinical observations, body temperature, body weight, food consumption, clinical pathology, immunogenicity, and post-mortem examinations including histopathology. Viral load, distribution, persistence, and shedding in tissues and body fluids were evaluated by quantitative reverse transcriptase polymerase chain reaction. RESULTS: The subcutaneous administration of CYD-TDV was well tolerated. There were no toxicological findings other than expected minor local reactions at the injection site. A transient low level of CYD-TDV viral RNA was detected in blood and the viral genome was identified primarily at the injection site and in the draining lymph nodes following immunization. CONCLUSIONS: These results, together with other data from repeat-dose toxicity and neurovirulence studies, confirm the absence of toxicological concern with CYD-TDV and corroborate clinical study observations.

Integrated immunogenicity analysis of a tetravalent dengue vaccine up to 4 y after vaccination.

Two large pivotal phase III studies demonstrated the efficacy of the tetravalent dengue vaccine (CYD-TDV; Dengvaxia®, Sanofi Pasteur) against all dengue serotypes. Here we present an unprecedented integrated summary of the immunogenicity of CYD-TDV to identify the parameters driving the neutralizing humoral immune response and evolution over time. We summarized the immunogenicity profiles of a 3-dose schedule of CYD-TDV administered 6 months apart across 10 phase II and 6 phase III trials undertaken in dengue endemic and non-endemic countries. Dengue neutralizing antibody titers in sera were determined at centralized laboratories using the 50% plaque reduction neutralization test (PRNT50) at baseline, 28 d after the third dose, and annually thereafter for up to 4 y after the third dose in some studies. CYD-TDV elicits neutralizing antibody responses against all 4 dengue serotypes; geometric mean titers (GMTs) increased from baseline to post-dose 3. GMTs were influenced by several parameters including age, baseline dengue seropositivity and region. In the 2 pivotal studies, GMTs decreased initially during the first 2 y post-dose 3 but appear to stabilize or slightly increase again in the third year. GMTs persisted 1.2-3.2-fold higher than baseline levels for up to 4 y post-dose 3 in other studies undertaken in dengue endemic countries. Our integrated analysis captures the fullness of the CYD-TDV immunogenicity profile across studies, age groups and regions; by presenting the available data in this way general trends and substantial outliers within each grouping can be easily identified. CYD-TDV elicits neutralizing antibody responses against all dengue serotypes, with differences by age and endemicity, which persist above baseline levels in endemic countries.