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Randomized Controlled Trial
. 2015 Jul;89(14):7348-62.
doi: 10.1128/JVI.00273-15.

Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies

Randomized Controlled Trial

Complexity of Neutralizing Antibodies against Multiple Dengue Virus Serotypes after Heterotypic Immunization and Secondary Infection Revealed by In-Depth Analysis of Cross-Reactive Antibodies

Wen-Yang Tsai et al. J Virol. 2015 Jul.

Abstract

The four serotypes of dengue virus (DENV) cause the most important and rapidly emerging arboviral diseases in humans. The recent phase 2b and 3 studies of a tetravalent dengue vaccine reported a moderate efficacy despite the presence of neutralizing antibodies, highlighting the need for a better understanding of neutralizing antibodies in polyclonal human sera. Certain type-specific (TS) antibodies were recently discovered to account for the monotypic neutralizing activity and protection after primary DENV infection. The nature of neutralizing antibodies after secondary DENV infection remains largely unknown. In this study, we examined sera from 10 vaccinees with well-documented exposure to first and second DENV serotypes through heterotypic immunization with live-attenuated vaccines. Higher serum IgG avidities to both exposed and nonexposed serotypes were found after secondary immunization than after primary immunization. Using a two-step depletion protocol to remove different anti-envelope antibodies, including group-reactive (GR) and complex-reactive (CR) antibodies separately, we found GR and CR antibodies together contributed to more than 50% of neutralizing activities against multiple serotypes after secondary immunization. Similar findings were demonstrated in patients after secondary infection. Anti-envelope antibodies recognizing previously exposed serotypes consisted of a large proportion of GR antibodies, CR antibodies, and a small proportion of TS antibodies, whereas those recognizing nonexposed serotypes consisted of GRand CR antibodies. These findings have implications for sequential heterotypic immunization or primary immunization of DENV-primed individuals as alternative strategies for DENV vaccination. The complexity of neutralizing antibodies after secondary infection provides new insights into the difficulty of their application as surrogates of protection.

Importance: The four serotypes of dengue virus (DENV) are the leading cause of arboviral diseases in humans. Despite the presence of neutralizing antibodies, a moderate efficacy was recently reported in phase 2b and 3 trials of a dengue vaccine; a better understanding of neutralizing antibodies in polyclonal human sera is urgently needed.We studied vaccinees who received heterotypic immunization of live-attenuated vaccines, as they were known to have received the first and second DENV serotype exposures.We found anti-envelope antibodies consist of group-reactive (GR), complex-reactive (CR), and type-specific (TS) antibodies, and that both GR and CR antibodies contribute significantly to multitypic neutralizing activities after secondary DENV immunization. These findings have implications for alternative strategies for DENV vaccination. Certain TS antibodies were recently discovered to contribute to the monotypic neutralizing activity and protection after primary DENV infection; our findings of the complexity of neutralizing activities after secondary immunization/infection provide new insights for neutralizing antibodies as surrogates of protection.

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Figures

FIG 1
FIG 1
Binding specificity and neutralizing activities of vaccinee sera. (A to F) Day 42 sera from three vaccinees (ID19 [A and B]; ID1 [C and D]; ID12 [E and F]) after primary and secondary immunizations were subjected to WB analysis at 1:500 dilution (A, C, and E) and FRNT titer determinations (B, D, and F). (G) Previously described mouse GR anti-E MAbs (FL0232, 4G2) (21) were used to verify that comparable amounts of DENV and WNV antigens were loaded. Data are means and standard deviations of duplicates from one of two experiments. WN, WNV; Mo, mock.
FIG 2
FIG 2
Contribution of GR anti-E Abs to neutralizing activities after secondary immunization. Day 42 sera from two vaccinees (ID22, A and B; ID6, C and D) after secondary immunization were depleted with WNV antigen or mock treatment to remove GR Abs, as described in Materials and Methods. Postdepletion sera were examined by WB analysis, DB assay, virion ELISA at a 1:2,000 dilution (A and C), and neutralizing titers (FRNT50) (B and D). A previously described mouse GR anti-E MAb (FL0232) (21) was used to verify that comparable amounts of DENV and WNV antigens were loaded for the WB analysis and DB assay (A, right). Data are means and standard deviations of two experiments, each with duplicates. WN, WNV; Mo, mock. *, P = 0.03, two-tailed Mann-Whitney test, comparing OD450 values (A and C) and relative FRNT50 titers (B and D) between WNV- and mock-depleted sera for each DENV serotype or WNV.
FIG 3
FIG 3
Epitopes and IgG avidities of eluted GR Abs after secondary immunization, compared with total serum IgG avidity after primary and secondary immunization. (A and B) Day 42 serum from vaccinee ID 20 after secondary DENV2 immunization was depleted with WNV antigen; GR anti-E Abs were eluted from an NC membrane as described in Materials and Methods and subjected to epitope mapping by DB assay (A) and capture ELISA using WT and mutant VLPs containing key FL mutations (B) (21). (C to E) Avidity indices of eluted GR anti-E Abs and total serum were determined as described in Materials and Methods. Vaccinees were subgrouped by sequence of immunization, including DENV4 → DENV1 (ID1, ID4, ID5, and ID6 [D]), DENV2 → DENV1 (ID11 and ID12 [E]), and DENV4 → DENV2 (ID16, ID19, and ID20 [F]). A pooled DENV-immune human serum was used to verify that comparable amounts of DENV were loaded in the virion ELISA (C). Data are means and standard deviations of duplicates from one of two experiments (A to C) or means and standard deviations of duplicates from two experiments for all individuals within each subgroup (D to E). The P values were determined by a two-tailed Mann-Whitney test.
FIG 4
FIG 4
Contribution of CR anti-E Abs to neutralizing activities after secondary immunization. (A to C) Day 42 serum from vaccinee ID 1 after secondary immunization was depleted with WNV antigen and WNV plus DENV3 antigens to remove GR Abs and GR plus CR Abs, respectively, as described in Materials and Methods. Depletion with mock and mock-mock sera served as controls. Postdepletion sera were examined by virion ELISA at a 1:2,000 dilution (A) and by FRNT (C). A human anti-E GR MAb (82.11LALA) was used to verify that comparable amounts of DENV and WNV antigens were loaded (B) (25, 44). Data are means and standard deviations from two experiments, each with duplicates. *, +, and x, P = 0.03, two-tailed Mann-Whitney test, comparing OD450 values between WNV- and mock-depleted sera (*) or between WNV-D3- and mock-mock-depleted sera (+) to each serotype or WNV (A), and comparing the relative FRNT50 between WNV-D3- and mock-mock-depleted sera (x) to each serotype (C). (D) Summary of the percent reduction in FRNT50 titer after depletion with WNV plus DENV3 antigens in four vaccinees. Data are means from two experiments, each with duplicates. The P value was determined by comparing the percent reduction in FRNT50 titers (all data points of 4 vaccinees) against each serotype between WNV-D3- and mock-mock-depleted or WNV-depleted sera (two-tailed Mann-Whitney test). WN, WNV; Mo, mock.
FIG 5
FIG 5
Composition of anti-E Abs in sera after secondary immunization. (A and B) Day 42 serum from vaccinee ID 1 after secondary (DENV4 → DENV1) immunization was depleted with WNV antigen and examined by virion ELISA at a 1:2,000 dilution (A). Fourfold serial dilutions of the WNV- and mock-depleted sera were subjected to virion ELISA of each serotype to determine the endpoint titers and percent GR Abs (B), as described in Materials and Methods. (C and D) WNV-depleted serum was further depleted with DENV3 (a nonexposed serotype) antigen and examined by virion ELISA at 1:2,000 (C, left graph) and 1:500 (C, right graph). Fourfold serial dilutions of the WNV-DENV3- and mock-mock-depleted sera were subjected to virion ELISA for DENV1 and DENV4 to determine the endpoint titers and percentages of TS Abs (D), as described in Materials and Methods. (E) Summary of the composition of anti-E Abs in sera of four vaccinees. The percent CR Abs was calculated as follows: 100 – percent GR Abs – percent TS Abs. * and +, P = 0.03 (two-tailed Mann-Whitney test) for OD450s between WNV- and mock-depleted sera (*) or between WNV-D3- and mock-mock-depleted sera (+) to each DENV serotype or WNV. Data are means and standard deviations of duplicates from two experiments. WN, WNV; Mo, mock.
FIG 6
FIG 6
Contribution of GR anti-E Abs to neutralizing activities in patients after secondary DENV infection. (A and B) Sera from patient ID 32 at 3 months after secondary DENV1 infection were depleted with WNV antigen or mock depleted. Postdepletion sera were examined by virion ELISA at a 1:2,000 dilution (A) and by FRNT (B). A mouse (FL0232) and human (82.11LALA) GR anti-E MAb were used to verify that comparable amounts of DENV and WNV antigens were loaded in the virion ELISA (25, 44) (C). Data are means and standard deviations of two or three experiments, each with duplicates. *, P = 0.002; **, P = 0.009; x, P = 0.03 (two-tailed Mann-Whitney test, comparing OD450 values [* and **]) (A) and relative FRNT50 (x) (B) between WNV- and mock-depleted sera to each serotype or WNV. (D) Summary of the percent reduction in FRNT50 titers against 4 serotypes after depletion with WNV antigen in three patients. Data are means from two experiments, each performed in duplicate. The P value was determined by comparing the percent reduction in FRNT50 titers (all data points of 3 patients) between WNV- and mock-depleted sera to each serotype (two-tailed Mann-Whitney test). (E) Fourfold serial dilutions of WNV- and mock-depleted sera were subjected to virion ELISA of each serotype to determine the endpoint titers (as described for Fig. 5). The percentages of GR Abs in three patients' sera after secondary DENV1 infection are summarized. Data are means of duplicates from one of two experiments. WN, WNV; Mo, mock.
FIG 7
FIG 7
Composition of anti-E Abs and contribution of different anti-E Abs to neutralizing activities in four vaccinees after secondary immunization. (A to D) The proportion of different anti-E Abs (percentages of GR, CR, and TS Abs) were based on data shown in Fig. 5E, and the contributions of GR and CR Abs to neutralizing activities are based on the percent reductions of FRNT50 titers in Table 2 and Fig. 4D for four vaccinees (ID1 [A], ID6 [B], ID19 [C], and ID20 [D]). After depletion of GR and CR Abs, the remaining neutralizing activities to previously nonexposed serotypes were designated undetermined (UD). (E) As a comparison, data from a previously reported case of primary DENV3 infection were summarized. Shown are the percentages of TS anti-E Abs, estimated based on endpoint titers in recombinant DIII and virion ELISAs (43), and their contribution to the monotypic neutralizing activity (27).

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