A Phase 1 Human Immunodeficiency Virus Vaccine Trial for Cross-Profiling the Kinetics of Serum and Mucosal Antibody Responses to CN54gp140 Modulated by Two Homologous Prime-Boost Vaccine Regimens

Abstract

A key aspect to finding an efficacious human immunodeficiency virus (HIV) vaccine is the optimization of vaccine schedules that can mediate the efficient maturation of protective immune responses. In the present study, we investigated the effect of alternate booster regimens on the immune responses to a candidate HIV-1 clade C CN54gp140 envelope protein, which was coadministered with the TLR4-agonist glucopyranosyl lipid A-aqueous formulation. Twelve study participants received a common three-dose intramuscular priming series followed by a final booster at either 6 or 12 months. The two homologous prime-boost regimens were well tolerated and induced CN54gp140-specific responses that were observed in both the systemic and mucosal compartments. Levels of vaccine-induced IgG-subclass antibodies correlated significantly with FcγR engagement, and both vaccine regimens were associated with strikingly similar patterns in antibody titer and FcγR-binding profiles. In both groups, identical changes in the antigen (Ag)-specific IgG-subclass fingerprint, leading to a decrease in IgG1 and an increase in IgG4 levels, were modulated by booster injections. Here, the dissection of immune profiles further supports the notion that prime-boost strategies are essential for the induction of diverse Ag-specific HIV-1 responses. The results reported here clearly demonstrate that identical responses were effectively and safely induced by both vaccine regimens, indicating that an accelerated 6-month regimen could be employed for the rapid induction of immune responses against CN54gp140 with no apparent impact on the overall quality of the induced immune response. (This study has been registered at http://ClinicalTrials.gov under registration no. NCT01966900.).

Keywords: IgG subclasses; adjuvant; homologous prime-boost strategy; human immunodeficiency virus envelope protein; human immunodeficiency virus vaccine; mucosal compartment; vaccine interval.

Figure 1

CONSORT Flow diagram. Numbers of participants recruited into the trial. Fifteen individuals were screened for the study, 14 were enrolled into the study (8 were enrolled into Group A, 6 into Group B), and 13 volunteers received all four intramuscular immunizations.

Figure 2

Antigen (Ag)-specific humoral responses in human serum samples following homologous prime-boost vaccination regimens. Participants received 3× priming immunizations (IMs) of CN54gp140 adjuvanted with GLA-AF on days 0, 28, and 56, followed by a booster injection at day 168 (Group A) or day 336 (Group B). Indicated by dotted line and numbers 1°–4°. (A,B) CN54gp140-specific IgG, IgA, IgM and (C,D) IgG-subclass concentrations were measured in human serum/plasma and expressed as mean ± SEM. (E) The sum of the four IgG subclasses was calculated and used to determine the percentage of CN54gp140-specific IgG-subclass antibodies 14 days after the priming phase (D70) and 14 days after the respective booster injections (D182/D350) for both Groups A and B. The proportion of Ag-specific IgG1 was decreased, while Ag-specific IgG4 levels were significantly increased after the respective booster IMs. Non-parametric Mann–Whitney test (*p < 0.05, **p < 0.01).

Figure 3

Antigen (Ag)-specific humoral responses in seminal and rectal secretions following homologous prime-boost immunization (IM). Participants received 3× priming IMs of CN54gp140 adjuvanted with GLA-AF on days 0, 28, and 56, followed by a booster injection at day 168 (Group A) or day 336 (Group B); indicated by dotted line and numbers 1°–4°. (A–D) CN54gp140-specific IgG was measured in human serum/plasma, seminal (n = 8) and rectal secretions (n = 11) and expressed as mean ± SEM. (E) Ag-specific IgG levels in seminal secretions correlate significantly with systemic IgG in both groups, while correlation coefficients (r-values) were lower when comparing Ag-specific IgG in rectal and systemic compartments. Spearman’s rank correlation coefficient (p < 0.0001).

Figure 4

Peptide array analysis of Env-specific IgG responses. (A,B) Specificity of serum IgG responses at 14 days after the third and fourth intramuscular immunization (IM) for Group A and B. Shown is the median fluorescence intensity (MFI). IgG responses against individual antigenic regions were considered positive if the corresponding fluorescence intensity was above 2,500 after subtraction of the prevaccination value (D0). The MFI was then calculated for region-specific IgG responses occurring in at least 25% of vaccines. (C) Comparison of total fluorescence intensity of ENV-specific serum antibodies 4 days post third/fourth IM for Group A and B showed no significant differences (Mann–Whitney test).

Figure 5

FcγR-binding profiles and breadth of antigen (Ag)-specific humoral responses in serum samples following homologous prime-boost immunization. The ability of vaccine-induced antibodies recognizing clade C.CN54gp140, A.UG37gp140, and D.UG21gp140 human immunodeficiency virus (HIV) proteins to bind dimeric (A,B) FcγRIIIa or (C,D) FcγRIIa was assessed in a customized multiplex dimer assay. (E,F) For further comparison, HIV-binding antibodies (E,F) were determined for the three selected clades for both study groups (A, n = 6 and B, n = 5).

Figure 6

Correlations between CN54gp140-specific IgG subclass measurements and FcγRIIa (CD32a)/FcγRIIIa (CD16a) binding profiles for Group A and B. A customized multiplex assay was used to determine binding of dimerized FcγRIIa or FcγRIII following binding to different human immunodeficiency virus (HIV)-clade proteins in serum samples from Group A and B. For simplicity, the horizontal and vertical labels, indicating the various HIV-clade proteins, were labeled with the ending .2a or .3a, respectively, to designate FcγR dimers. Correlations between CN54gp140-specific IgG subclasses are boxed in red, and correlations between FcγRIIa/FcγRIIIa binding profiles are boxed in blue, while correlations directly related to the CN54gp140 antigen, which was used for serial immunizations, are boxed in green. C.CN54gp140-mediated engagement of both FcγRII- and FcγRIIIa-receptors was positively associated with CN54gp140-specific IgG1/2 in both groups. Both study groups exhibit identical isotypic and FcγR-binding profiles. r_S—Spearman’s rank correlation coefficient (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7

Analysis of the memory B cell (mBC) response induced by sequential immunization with the human immunodeficiency virus-antigen CN54gp140. Frozen peripheral blood mononuclear cells (PBMCs) were thawed and expanded using human IL-2 and R848 for 4 days. Expanded cells were assayed for antigen (Ag)-specific and total IgG-secreting ASCs by ELISPOT assay at 0, 84, and 196 or 364 days, respectively. Each sample was measured in triplicate, averaged and plotted as Ag-specific per total mBC. (A) Percentage of Ag-specific mBC per total IgG-secreting mBC. Due to the limitation in availability of PBMC at least one data point is missing for the following participants: S001 (D0), S002 (D0), and S013 (D0 and D196). (B) Ag-specific spot forming units/well (indicated by lower indices) Ag-specific mBC and total-IgG-secreting mBC for one selected donor from each group for days 0, 84, and 196 or 364, respectively.