Comparative evaluation of three assays for measurement of dengue virus neutralizing antibodies.

Plaque reduction neutralization tests (PRNTs) are commonly used for measuring levels of dengue virus (DENV) neutralizing antibodies. However, these assays lack a standardized format, generally have a low sample throughput, and are labor-intensive. The objective of the present study was to evaluate two alternative DENV neutralizing antibody assays: an enzyme-linked immunosorbent assay-based microneutralization (MN) assay, and a fluorescent antibody cell sorter-based, DC-SIGN expresser dendritic cell (DC) assay. False-positive rates, serotype specificity, reproducibility, sensitivity, and agreement among the assay methods were assessed using well-characterized but limited numbers of coded test sera. Results showed that all three assays had false-positive rates of less than 10% with titers near the cut-off and generally below the estimated limits of detection. All three methods demonstrated a high degree of specificity and good agreement when used to assay sera and serum mixtures from monovalent vaccinees and sera from patients after primary natural infection, with the only notable exception being moderate-to-high neutralizing antibody titers against DENV 2 measured by PRNT in a mixture containing only DENV 3 and DENV 4 sera. The MN and DC assays demonstrated good reproducibility. All three assays were comparable in their sensitivity, except that the PRNT was less sensitive for measuring DENV 4 antibody, and the MN and DC assays were less sensitive for measuring DENV 2 antibody. However, when used to test sera from persons after tetravalent DENV vaccination or secondary DENV infection, there was poor specificity and poor agreement among the different assays.

An evaluation of dengue type-2 inactivated, recombinant subunit, and live-attenuated vaccine candidates in the rhesus macaque model.

The safety, immunogenicity, and protective efficacy of two non-replicating antigen-based vaccines and one live-attenuated virus (LAV) vaccine for dengue type-2 (dengue-2) virus were evaluated in the rhesus macaque model. The non-replicating vaccines consisted of whole, purified inactivated virus (PIV) and a recombinant subunit protein containing the amino-(N)-terminal 80% of envelope protein (r80E), each formulated with one of five different adjuvants. Each formulation was administered to three animals on a 0, 3-month schedule. Following the primary immunizations, 37 of 39 animals demonstrated dengue-2 virus neutralizing antibodies. After the booster immunizations all animals had dengue neutralizing antibodies with peak titers ranging from 1:100 to 1:9700. The highest neutralizing antibody titers were observed in the groups that received r80E antigen formulated with AS04, AS05, or AS08 adjuvant, and PIV formulated with AS05 or AS08 adjuvant. These newer adjuvants are based on alum, fraction QS-21 of saponin, and monophosphoryl lipid A (MPL). Protection was evaluated by dengue-2 virus challenge 2 months after the booster by the measurement of circulating virus (viremia) and post-challenge immune responses. Several groups exhibited nearly complete protection against viremia by bioassay, although there was evidence for challenge virus replication by Taqmantrade mark and immunological assays. None of the vaccines conferred sterile immunity.

Immunologic analysis of a phase I/II study of vaccination with MAGE-3 protein combined with the AS02B adjuvant in patients with MAGE-3-positive tumors.

In a phase I/II study, patients with solid metastatic MAGE-3-positive tumors, mainly melanoma, were vaccinated with recombinant MAGE-3 protein combined with the immunologic adjuvant AS02B comprised of MPL and QS21 in an oil-in-water emulsion. The recombinant MAGE-3 protein was made up of a partial sequence of the protein D (ProtD) antigen of Haemophilus influenzae fused to the MAGE-3 sequence. The vaccine was given intramuscularly at 3-week intervals. Patients whose tumors stabilized or regressed after 4 vaccinations received 2 additional vaccinations at 6-week intervals. MAGE-3 and ProtD antibody and cellular immune responses were monitored after vaccination. Ninety-six percent (23/24) of the patients vaccinated with MAGE-3 protein in AS02B adjuvant elicited a significant anti-MAGE-3 IgG antibody response after 4 vaccinations, and all developed anti-ProtD IgG antibodies. For the detection of T-cell activity, total peripheral blood mononuclear cells were restimulated in vitro with MAGE-3- or ProtD-loaded autologous mature dendritic cells. In 30% of the evaluable patients vaccinated with the adjuvanted recombinant protein, IFNgamma production was increased in response to MAGE-3, and 2 patients (14% of evaluable patients) had a concomitant increase in IL-5 production. In 37% and 43% of the patients, respectively, IFNgamma or IL-5 production was increased in response to ProtD. It is concluded that vaccination of advanced cancer patients with MAGE-3 self-antigen in AS02B adjuvant is able to elicit MAGE-3-specific antibody and a T-cell response.