A tetravalent live attenuated dengue virus vaccine stimulates balanced immunity to multiple serotypes in humans

The four-dengue virus (DENV) serotypes infect several hundred million people annually. For the greatest safety and efficacy, tetravalent DENV vaccines are designed to stimulate balanced protective immunity to all four serotypes. However, this has been difficult to achieve. Clinical trials with a leading vaccine demonstrated that unbalanced replication and immunodominance of one vaccine component over others can lead to low efficacy and vaccine enhanced severe disease. The Laboratory of Infectious Diseases at the National Institutes of Health has developed a live attenuated tetravalent DENV vaccine (TV003), which is currently being tested in phase 3 clinical trials. Here we report, our study to determine if TV003 stimulate balanced and serotype-specific (TS) neutralizing antibody (nAb) responses to each serotype. Serum samples from twenty-one dengue-naive individuals participated under study protocol CIR287 (ClinicalTrials.gov NCT02021968) are analyzed 6 months after vaccination. Most subjects (76%) develop TS nAbs to 3 or 4 DENV serotypes, indicating immunity is induced by each vaccine component. Vaccine-induced TS nAbs map to epitopes known to be targets of nAbs in people infected with wild type DENVs. Following challenge with a partially attenuated strain of DENV2, all 21 subjects are protected from the efficacy endpoints. However, some vaccinated individuals develop post challenge nAb boost, while others mount post-challenge antibody responses that are consistent with sterilizing immunity. TV003 vaccine induced DENV2 TS nAbs are associated with sterilizing immunity. Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development.

This is a sophisticated study on the attributes of neutralizing antibodies circulating 6 months after vaccination of 21 seronegative adults with a single dose of a tetravalent live-attenuated dengue vaccine. This is followed by a further study of the anamnestic neutralizing antibody responses observed after challenge with a single dose of live-attenuated dengue 2 virus. Based upon absorption tests, after a single dose of vaccine 13 DENV 1; 16 DENV 2; 18 DENV 3 and all 21 DENV 4 vaccinees circulated type specific neutralizing antibodies. Using novel genetic transplant methods the authors determined that antibodies bearing wild-type DENV neutralizing epitopes for each of the 4 DENVs circulated after a single dose of tetravalent vaccine. Novel to this experiment is the detection of low levels of IgM DENV antibodies post live virus challenge in a small number of subjects in the absence of viremia. Some, but not all had low levels of DENV 1 type specific neutralizing antibodies prior to challenge. This raises the question of whether there might have been some replication of the DENV 2 challenge virus. Specific comments: Line 54. The final sentence needs to be stated correctly. Firstly, the word "correlate" appears twice. Importantly, without vaccine test results from a phase 3 study, it is not possible to generalize to the conclusion that the vaccine under study is protective. The word "may" should be introduced. Line 88. The statement in this sentence is an observation. There is no evidence linking unbalanced replication of vaccine viruses to failure of Dengvaxia to protect against dengue virus infection or to explain enhanced disease in vaccinated seronegatives. Supplement: What is the reason why the PRNT data for dengue 2, column 2 in table S 1 differ from data in column 1 in table S 4? Table S 4: The authors observed a 3 -8-fold increase in DENV 2 neutralizing antibody titers in 5 vaccinees on day 270, 90 days after administering challenge virus. The reviewer wonders if the boost observed in DENV 2 PRNT might be heterotypic antibodies related to low levels of DENV 1 PRNT in sera # 2 and 3, for example? Were boosts observed in DENV 1 PRNTs after challenge? It is not clear when and how long after challenge the IgM antibodies were detected. Are the authors aware that a rapid decline in anamnestic antibody titers was observed in monotypic dengue-immune monkeys challenged with homologous virus while heterologous challenged animals produced a sustained elevation of antibody responses?1 It would be interesting to know the PRNT titers in these animals 6 months after challenge. (a tetravalent dengue virus vaccine candidate). The authors aimed to assess whether TV003 elicited balanced and serotype-specific neutralizing antibody responses to all 4 dengue virus serotypes. They demonstrate that immunization with TV003 resulted in neutralizing antibody production against 3 or 4 serotypes in 76% of the volunteers and that following challenge with an attenuated dengue virus 2 strain, 16/21 volunteers had sterilizing immunity against DENV-2.
Major comments: Figure 1 and corresponding text indicate that only DENV-2 depletion was performed to measure TS nAb against DENV-1, 3, and 4. In the Methods section (lines 421-423), it states that "A protocol using a different depletion condition... would be expected to yield similar results". Please provide data or a reference to support this claim.

Reviewer#1:
• Line 54. The final sentence needs to be stated correctly. Firstly, the word "correlate" appears twice. Importantly, without vaccine test results from a phase 3 study, it is not possible to generalize to the conclusion that the vaccine under study is protective. The word "may" should be introduced.

Response:
We agree with the reviewer and now have removed the word "correlate" twice and introduce the word "may" as suggested. The changes made are highlighted in the main text of the manuscript in lines 54-56 on page 3. The new sentence is rewritten as below.
"Our results indicate that nAbs to TS epitopes on each serotype may be a better correlate than total levels of nAbs currently used for guiding DENV vaccine development".
• Line 88. The statement in this sentence is an observation. There is no evidence linking unbalanced replication of vaccine viruses to failure of Dengvaxia to protect against dengue virus infection or to explain enhanced disease in vaccinated sero negatives.

Response:
We disagree with the reviewer's comment that there is no evidence linking unbalanced replication to vaccine efficacy. For Dengvaxia, several publications have established that the DENV4 component replicates to higher levels than the other three components in humans and animals (Guirakhoo et.al., Virology, 2002;Guy et.al., Am J Trop Med Hyg, 2009;Osorio et.al., Am J Trop Med Hyg, 2011;Morrison et.al., J Infect Dis, 2010;Dayan et.al., Hum Vaccin Immuother, 2014;Torresi et.al., J Infect Dis, 2017). This replication pattern is correlated with most baseline seronegative subjects developing DENV4 TS nAbs and lower levels of DENV1, 2 and 3 CR nAbs after receiving Dengvaxia (Henein et.al., J Infect Dis, 2017). Finally, in Sanofi's clinical trials, while overall vaccine efficacy was low, serotype-specific efficacy against DENV4 was high even after 5-6 years of follow up (Sridhar et. al. N Engl J Med, 2018;and Gustavo et.al., Vaccine, 2020

Supplement:
• What is the reason why the PRNT data for dengue 2, column 2 in table S1 differ from data in column 1 in • Table S4: The authors observed a 3-8-fold increase in DENV2 neutralizing antibody titers in 5 vaccinees on day 270, 90 days after administering challenge virus. The reviewer wonders if the boost observed in DENV 2 PRNT might be heterotypic antibodies related to low levels of DENV 1 PRNT in sera # 2 and 3, for example? Were boosts observed in DENV 1 PRNTs after challenge? It is not clear when and how long after challenge the IgM antibodies were detected. Are the authors aware that a rapid decline in anamnestic antibody titers was observed in monotypic dengue-immune monkeys challenged with homologous virus while heterologous challenged animals produced a sustained elevation of antibody responses? It would be interesting to know the PRNT titers in these animals 6 months after challenge.

Major comments
• Figure 1 and corresponding text indicate that only DENV-2 depletion was performed to measure TS nAb against DENV-1, 3, and 4. In the Methods section (lines 421-423), it states that "A protocol using a different depletion condition... would be expected to yield similar results". Please provide data or a reference to support this claim.

Response:
We have provided below, data supporting the claim that "a different strategy or protocol to deplete the sera would be expected to yield similar results" (for example a DENV1 depleted sera to estimate TS nAb against DENV2, 3, and 4 yield similar results)".