Edited by: Gabriella Scarlatti, San Raffaele Hospital (IRCCS), Italy
Reviewed by: Christiane Moog, Institut National de la Santé et de la Recherche Médicale (INSERM), France; Cynthia Ann Derdeyn, Emory University, United States
This article was submitted to Comparative Immunology, a section of the journal Frontiers in Immunology
†These authors have contributed equally to this work
‡Present Address: Marzena Pazgier, Infectious Disease Division of Department of Medicine of Uniformed Services of University of the Health Sciences, Bethesda, MD, United States
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Recent clinical trials and studies using nonhuman primates (NHPs) suggest that antibody-mediated protection against HIV-1 will require α-HIV envelope humoral immunity beyond direct neutralization to include Fc-receptor (FcR) mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC). There is also strong evidence indicating that the most potent ADCC response in humans is directed toward transitional non-neutralizing epitopes associated with the gp41-interactive face of gp120, particularly those within the first and second constant (C1–C2) region (A32-like epitopes). These epitopes were shown to be major targets of ADCC responses during natural infection and have been implicated in vaccine-induced protective immunity. Here we describe the immunogenicity of ID2, an immunogen consisting of the inner domain of the clade A/E 93TH057 HIV-1 gp120 expressed independently of the outer domain (OD) and stabilized in the CD4-bound conformation to harbor conformational A32 region epitopes within a minimal structural unit of HIV-1 Env. ID2 induced A32-specific antibody responses in BALB/c mice when injected alone or in the presence of the adjuvants Alum or GLA-SE. Low α-ID2 titers were detected in mice immunized with ID2 alone whereas robust responses were observed with ID2 plus adjuvant, with the greatest ID2 and A32-specific titers observed in the GLA-SE group. Only sera from groups immunized in the presence of GLA-SE were capable of mediating significant ADCC using NKr cells sensitized with recombinant BaL gp120 as targets and human PBMCs as effectors. A neutralization response to a tier 2 virus was not observed. Altogether, our studies demonstrate that ID2 is highly immunogenic and elicits A32-specific ADCC responses in an animal host. The ID2 immunogen has significant translational value as it can be used in challenge studies to evaluate the role of non-neutralizing antibodies directed at the A32 subregion in HIV-1 protection.
The design of immunogens which induce broadly protective antibody responses against human immunodeficiency virus type 1 (HIV-1) is a major goal of HIV-1 vaccine development. This goal is formidable as HIV-1 evades immune surveillance via a number of escape mechanisms (
By contrast, less is known about mechanisms of vaccine induced humoral responses that act solely through Fc-mediated effector functions, including antibody-dependent cell-mediated cytotoxicity (ADCC). Epitopes involved solely in Fc-mediated processes are usually exposed late during viral entry and are thus targeted by antibodies that lack direct neutralizing activity. One group of these potent ADCC targets constitute the CD4-inducible (CD4i) epitopes within the gp120 molecule, referred to as Cluster A epitopes (
Here we describe the immunogenicity of a gp120 sub domain immunogen, referred to as ID2, designed by our group to stimulate humoral responses involving solely FcR-effector mechanisms designed to elicit an ADCC response in the absence of a neutralizing response (
A HEK 293 cell line stably expressing the ID2 immunogen was generated using the plasmid previously used for transient protein production, as in (
BALB/c mice were purchased from The Jackson Laboratory and housed in the animal facility managed by BIOQUAL's, Inc., Rockville, MD. The mice were cared for in accordance with the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC) standards and all procedures involving animals were approved by the University Committee on Use and Care of Animals (UCUCA) of BIOQUAL, Inc. 6–8 weeks old BALB/c mice (male and female, 6 animals per group) were immunized at week 0, 2, 4, and 8 via IP injections of 20 μg of ID2 protein in different adjuvants. The control group received ID2 immunogen in PBS and two adjuvants were also trialed; ID2 immunogen in Alum (2% aluminum hydroxide wet gel suspension, InvivoGen, Catalog # vac-alu-250), and ID2 immunogen in GLA-SE adjuvant (stable oil-in water emulsion containing TLR-4 agonist developed by Infectious Disease Research Institute, Catalog # IDRI-GLA-SE, known also under the name EM082). Serum samples were collected prior to immunization and 2 weeks after each immunization according to the scheme shown in
ID2 immunogen design and immunization scheme.
The presence and titers of total IgG, IgG1, IgG2a, IgG2b or IgG2c, IgG3, IgA, and IgM antibodies, specific for ID2 recombinant protein in sera of immunized mice were determined by an Enzyme Linked Immunosorbent Assay (ELISA) using a 100-μL-per-well volume format. Blocking Buffer (Tris-buffered saline (TBS; 10 mM Tris and 100 mM NaCl; pH 8.0) with 5% no fat dry milk and 0.1% Nonidet P-40) was used as blocking solution and as diluting solution for sera and detecting Abs. TBS-T buffer (TBS with 0.1% Tween-20) was used as washing solution. ELISAs were performed as follows: ID2 recombinant protein (0.5 μg/ml) was adsorbed onto ELISA plates (Immunoblot 2HB Thermo, Milford, MA) overnight at 4°C. The plates were washed three times and incubated with 100 μl blocking buffer per well for 2 h at room temperature. Serially diluted sera, beginning at 1:100, were then added and allowed to react with the coated antigen for 2 h at 37°C. Sera was removed, the plates washed, and alkaline phosphatase-conjugated goat anti-mouse IgG (Sigma cat#A3562), IgG1, IgG2a, IgG2b, IgG2c, and IgG3 (SouthernBiotech cat# 1071-04, 1081-04, 1091-04, 1078-04, 1103-04, respectively), IgA and IgM antibodies (SouthernBiotech cat #1040-04 and 1021-04, respectively) diluted 1:1000 in blocking buffer, were added followed by incubation for 1 h at 37°C. After removal of unbound antibody and washing, the Blue Phos Microwell Phosphatase Substrate System (KPL 50–88-00) was used as a substrate to quantitate bound antibody. After 15 min incubation at room temperature, the reaction was stopped using APstop Solution (KPL 50-89-00) and the optical density was read on a microplate reader (SpectraMax Paradigm Multi-Mode Detection Platform Molecular Devices) at 620 nm. The anti-ID2 antibody half-max binding serum titer was calculated using a Microsoft Excel iteration formula.
To determine if the immunization with ID2 immunogen had induced Cluster A like serum Abs, sera from the terminal bleed were tested for their ability to compete with Cluster A mAbs (A32, N5-i5) for the binding to ID2 in an ELISA setting. Plates were coated o/n at 4°C with 0.5 μg/ml of ID2. Ten-fold serial dilutions of immune sera and pre-immunization sera were mixed 1:1 with biotinylated human CD4i anti-envelope mAb at a concentration correspondent to the half max binding concentrations for each tested mAb; 0.66 μg/mL of A32 and 0.2 μg/ml of N5-i5. The mixtures were then added to previously washed and blocked plates. As a control, a 10-fold dilution of unbiotinylated CD4i mAbs A32 and N5-i5, starting at 10 μg/mL, were tested in the same assay. Sera and Abs were prepared as 2x solutions. After 2 h incubation at r.t. assay wells were washed and incubated with avidin-AP (Invitrogen 1:1,000 dilution) and then with the Blue Phos Microwell Phosphatase Substrate System. Biotinylated-mAb binding was determined by measuring absorbance at 620 nm. Competition percentage was calculated using GraphPad Prism as follows: 0% inhibition was defined as the mean OD value of the lowest serial dilution of the pre-immunization sera (1:106) while 100% inhibition was defined as the mean OD value of the highest concentration tested for unbiotinylated mAbs A32 and N5-i5 (10 μg/ml).
To assess if immunization with ID2 recombinant protein elicited serum antibodies recognizing conformational epitopes on ID2, pooled sera collected 2 weeks after the last immunization were incubated in solution o/n at 4°C with 1 μg/mL of denatured ID2. ID2 recombinant protein denaturation was performed as previously described in Moore et al. (
The ADCC activity of immunoglobulins present in mouse sera collected at week 10 were tested with the optimized rapid fluorometric antibody-dependent cellular cytotoxicity (RFADCC) assay (
Mouse sera collected 2 weeks after the 4th immunization and pre-immune sera, were tested in a TZM-bl assay for the presence of neutralizing antibodies. Briefly, 3-fold serial dilution (starting from 1/100 dilution) of sera were mixed with JR-FL pseudo-virus (TCID of 45000 Relative Luminescence Unit) and incubated at 37°C in 5% CO2 atmosphere for 60 min at room temperature. TZM-bl cells (10,000/well in complete RPMI with 11 μg/mL DEAE-Dextran) were then added to the sera-pseudo virus mix and plates were incubated for 48 h at 37°C in 5% CO2 atmosphere. One hundred and Fifty Microliter of supernatant were removed and 100 μL/well of BrightGlo was then added to each well and after a 2 min incubation to allow complete cell lysis, 100μL from each well was transferred to 96 well black plates. Plates were read with a luminometer using the Promega BrightGlo program. Percent neutralization was determined by calculating the difference in average relative luminescence units (RLU) between virus control (no serum/antibody) and test wells (cells + serum or antibody sample + virus), dividing this result by the difference in average RLU between virus control (cell + virus) and cell only wells, and multiplying by 100.
Differences in responses between ID2 alone, ID2+Alum, and ID2+GLA-SE were analyzed using a Two-way ANOVA with Bonferroni post-test comparing every sample to every other sample. All statistical analysis was carried out using GraphPad Prism (Version 5 for Windows, San Diego, CA, USA). ****represents statistical significance of
ID2 was designed to stably present the conformational CD4-inducable epitopes of the A32 region within a minimal structural unit of gp120 without any other known (neutralizing or non-neutralizing) epitopes present (
To evaluate if ID2 indeed is capable of selective induction of humoral response to the desired A32 region we performed immunogenicity studies using the recombinant preparation of ID2 obtained by mammalian cell culture (to preserve wild-type glycosylation) and BALB/c mice as an animal host. Purified ID2 protein, 20 μg per injection, was used to immunize groups of mice (6 animals per group) in the absence of adjuvant (in PBS) and with two adjuvant choices; Alum (2% aluminum hydroxide wet gel suspension) and GLA-SE (a stable oil-in water emulsion containing TLR-4 agonist adjuvant developed by Infectious Disease Research Institute). Immunizations were done according to the immunization scheme shown in
Sera collected 2 weeks after each immunization were tested in a sandwich ELISA to assess the presence of specific anti-ID2 serum antibodies. As shown in
Kinetics and titers of anti-ID2 immune response in immunized mice. Sera collected 2 weeks after each immunization were tested in ELISA for the presence of anti-ID2 specific immune responses (total αID2 IgG). Each sample was assayed in duplicate, displayed is the Mean ± SEM of the 6 mice in each group for
By design ID2 consists of two faces; a face that harbors the conformational epitopes of the A32 region and a face that is exposed by the removal of the OD which does not have any known epitope targets. Although the epitopes within the newly exposed face are not known, this face might harbor epitopes that are rendered immunodominant by their exposure. To assess if the antibody response induced by ID2 is indeed specific for the desired A32 epitope region we tested sera collected after the final immunization in a competition ELISA with mAb A32 and the A32-like antibody N5-i5 (
Epitope and conformational specificity of serum antibodies elicited in immunized mice. Sera collected 2 weeks after the final immunization (week 10) were tested for their ability to compete with Cluster A biotinylated mAbs for the binding to ID2 recombinant protein in ELISA. Plates were coated with 0.5 μg/mL of ID2 and serum was incubated with
Next, we asked if the elicited antibody responses were directed toward conformational or linear epitopes within the ID2 immunogen. Pooled sera collected after the final immunization were incubated in solution with denatured ID2 (dID2) protein and then probed in ELISAs with non-denatured ID2 immunogen coated on microplates. As shown in
The ID2 immunogen was designed to harbor A32 or A32-like epitopes involved in potent ADCC responses against target cells during the earliest stage of viral entry i.e., at the interaction of gp120 of the Env trimer with the host cell receptor CD4 (
ADCC in sera from mice immunized with ID2 with and without adjuvant.
ID2 was designed specifically to contain no known neutralizing epitope targets (
Neutralization in sera from mice immunized with ID2 with and without adjuvant. Sera were tested in a TZM-bl assay for the presence of neutralizing activities against clade B Tier 2 JRFL pseudo-virus. Sera from Pre-bleed (Hollow circle), ID2 Alone (Black square), ID2 + Alum (Blue square), and ID2 + GLA-SE (Red square) were diluted and assayed for their ability to neutralize a clade B Tier 2 JR pseudo-virus (Bottom X axis). These samples were run alongside diluted A32 antibody (orange square) for a negative control and P7 antibody (green square) as a positive control, beginning at 10 μg/ml (Top X axis).
In addition to showing that ADCC in the absence of robust neutralization was adequate for protection against HIV, the RV144 trial also highlighted the importance of the type of antibody repertoire raised. A strong IgG response in the absence of IgA conferred the best protection (
Evaluation of IgG subclasses, IgA and IgM elicited in immunized mice. 10-fold dilutions of pre-immunized (pre-bleed, Black circle) and immunized mice sera collected after last immunization (week 10), were evaluated in ELISA to assess the IgG subtypes;
Antibodies capable of effective Fc-mediated effector functions, including ADCC, have recently received increasing interest as important components of a vaccine induced humoral response. Although the enthusiasm for this type of antibody function was mostly evoked by the RV144 trial, the evidence also exists from vaccination strategies with Env immunogens in non-human primates (NHP) that link FcR effector functions of antibodies with post-infection control of viremia and/or blocking HIV-1 acquisition, often in the absence of neutralization (
Correlate analyses of the infection risk in the RV144 trial have indicated two gp120 epitope regions; the conformational C1-C2 and the linear V2 loop epitopes, as the major players involved in the Fc-mediated protective response (
Similarly, antibodies specific for Cluster A epitope region (including the A32 subregion) and the co-receptor binding site correlated with sterilizing heterologous protection against SHIV162p3 in NHPs immunized with the conformationally constrained gp120 immunogen, full-length single chain (FLSC) (
In this study, we investigated the immunogenicity and ability to induce an effective cross-clade ADCC response of a new immunogen candidate ID2, developed previously in our laboratory, as a minimal structural unit of HIV Env stably presenting the non-neutralizing epitopes within the A32 region (C1–C2 epitopes). ID2 consists of the inner domain construct stabilized in CD4-bound conformation by C65-C115 disulfide bond preserving the A32 region epitopes in the context of CD4-triggered full length gp120, derived from a clade A/E strain, without the complication of any other known neutralizing epitopes. We immunized BALB/c mice intraperitoneally (IP) with 4 doses of ID2 (20 μg /dose) over a period of 8 weeks with three immunizations with 2 weeks intervals and one final immunization delivered 4 weeks later to assess the induction of a memory antibody response. ID2 was administered either alone or with an adjuvant: Alum or GLA-SE. The resulting immune sera were evaluated for the presence of anti-ID2 antibodies (total IgG, IgG subclasses, IgA, and IgM) as well as for their capacity to bind to conformational A32 region epitopes and compete for the binding with mAbs specific for those epitopes. Immune sera were also tested in our RFADCC assay for their ability to mediate ADCC with CEM-NKr-CCR5 target cells sensitized with recombinant gp120 from the Clade B HIV-1BaL isolate and to neutralize a Tier 2 strain of HIV-1. Our results showed that immunization with ID2 alone elicited no or very low anti-ID2 serum antibodies and those elicited were only after the 4th immunization, whereas mice in the groups immunized with ID2 in adjuvants showed robust anti-ID2 responses. Of the two adjuvants, mice immunized with ID2 in GLA-SE exhibited higher anti-ID2 titers at the end of study (week 10) as compared with the Alum group. Interestingly, both groups showed comparable titers after the 3rd immunization, but while in the GLA-SE group the 4th immunization boosted the immune response significantly above the alum group, mice in the Alum group presented a comparable titer to week 6. These data indicate that administration of ID2 in combination with GLA-SE induces enhanced and easily boosted responses to the target antigen. This agrees with the data regarding GLA-SE as an adjuvant, which was designed to promote strong and long lasting TH1 responses to protein vaccine antigens (
ADCC was only significantly increased over the negative control in sera isolated from the GLA-SE adjuvant group, indicating that high titers of ID2-specific mouse IgG1, IgG2a, and IgG2b in addition to small amounts of other isotypes are required for effective RFADCC. In addition to generating more diverse isotypes of ID2 specific antibodies, immunizing with GLA-SE also led to the generation of more specific antibodies – as indicated by significantly higher percent competition for both A32 and N5-i5 antibodies. Interestingly, some sera displayed high ADCC yet low levels of ID2-specific IgG and an absence of class switching from IgG1. One example of this is sample 4,007; when looking at the individual data for the competition ELISA, 4,007 sera inhibits the binding of A32 by 39.7% at the highest sera concentration and N5-i5 by 45%, an average amount for A32 and below average for N5-i5 when compared to other mice in the Alum group. One possibility for a more potent ADCC response is that antibodies to different epitopes were raised in this particular mouse. While the design of ID2 prevents the elicitation of classical C11-like antibodies, some antibodies may be specific for C11-like epitopes which would not have been identified by the competition ELISAs carried out. One known examples of an antibody which could be raised to this area of ID2 is JR4 (
In conclusion, these results indicate that ID2 can be highly immunogenic, especially when administered together with GLA-SE as an adjuvant, and that the elicited immune response can mediate ADCC in a RFADCC assay using human PBMCs as effector cells. None of the immune sera showed robust neutralization activity against the tier 2 JRFL virus indicating that ID2 as an immunogen induces mostly a humoral immune response against the desired non-neutralizing epitopes within the A32 region. ID2 in combination with GLA-SE was successfully utilized to demonstrate that Fc-effector mechanisms in the absence of strong neutralizing antibodies could be directed toward the A32 subregion which has the potential for HIV-1 protection and ID2 could therefore be utilized for future studies in a NHP challenge study.
All datasets generated for this study are included in the manuscript and/or the supplementary files.
MV, NG, WT, and MP designed, performed research, and analyzed the data. AD, CO, RF, and GL carried out assays and analyzed the ADCC data. RS analyzed the data. MV, RS, and MP wrote the paper. All authors read the manuscript and provided comments or revisions.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
We thank our IHV colleagues for outstanding support of the studies leading to the ideas presented above. This work was supported by NIH Grants: NIAID R01 AI116274 to MP, R01AI129769 to MP and Andres Finzi and NIAID P01 AI120756 to Georgia Tomaras.