A Vaccine Encoding Conserved Promiscuous HIV CD4 Epitopes Induces Broad T Cell Responses in Mice Transgenic to Multiple Common HLA Class II Molecules

Current HIV vaccine approaches are focused on immunogens encoding whole HIV antigenic proteins that mainly elicit cytotoxic CD8+ responses. Mounting evidence points toward a critical role for CD4+ T cells in the control of immunodeficiency virus replication, probably due to cognate help. Vaccine-induced CD4+ T cell responses might, therefore, have a protective effect in HIV replication. In addition, successful vaccines may have to elicit responses to multiple epitopes in a high proportion of vaccinees, to match the highly variable circulating strains of HIV. Using rational vaccine design, we developed a DNA vaccine encoding 18 algorithm-selected conserved, “promiscuous” (multiple HLA-DR-binding) B-subtype HIV CD4 epitopes - previously found to be frequently recognized by HIV-infected patients. We assessed the ability of the vaccine to induce broad T cell responses in the context of multiple HLA class II molecules using different strains of HLA class II- transgenic mice (-DR2, -DR4, -DQ6 and -DQ8). Mice displayed CD4+ and CD8+ T cell responses of significant breadth and magnitude, and 16 out of the 18 encoded epitopes were recognized. By virtue of inducing broad responses against conserved CD4+ T cell epitopes that can be recognized in the context of widely diverse, common HLA class II alleles, this vaccine concept may cope both with HIV genetic variability and increased population coverage. The vaccine may thus be a source of cognate help for HIV-specific CD8+ T cells elicited by conventional immunogens, in a wide proportion of vaccinees.     

TLR4 Ligands Augment Antigen-Specific CD8+ T Lymphocyte Responses Elicited by a Viral Vaccine Vector

Toll-like receptor (TLR) ligands are critical activators of innate immunity and are being developed as vaccine adjuvants. However, their utility in conjunction with viral vector-based vaccines remains unclear. In this study, we evaluated the impact of a variety of TLR ligands on antigen-specific CD8⁺ T lymphocyte responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector expressing simian immunodeficiency virus Gag in mice. The TLR3 ligand poly(I:C) suppressed Gag-specific cellular immune responses, whereas the TLR4 ligands lipopolysaccharide and monophosphoryl lipid A substantially augmented the magnitude and functionality of these responses by a MyD88- and TRIF-dependent mechanism. These data demonstrate that TLR ligands can modulate the immunogenicity of viral vaccine vectors both positively and negatively. Moreover, these findings suggest the potential utility of TLR4 ligands as adjuvants for rAd vector-based vaccines.Toll-like receptors (TLRs) are critical sensors of infection with a fundamental role in the activation of innate immune responses and the subsequent modulation of pathogen-specific adaptive immunity (2). TLR ligands have therefore emerged as potential vaccine adjuvants, particularly in the context of peptide, protein, and DNA vaccines (17). In particular, TLR agonists are widely reported to modulate antibody and T helper lymphocyte responses, and in some cases CD8⁺ T lymphocyte responses, elicited by protein-based vaccines (5, 19, 33, 41). However, far less is known about the impact of TLR ligands on the immunogenicity of viral vector-based vaccines.Compared with DNA vaccines, viral vectors are typically more immunogenic, presumably as a result of the activation of innate immunity via multiple TLRs or other pattern recognition receptors (29). Viral vectors elicit robust T lymphocyte responses and thus are attractive vaccine candidates for pathogens such as human immunodeficiency virus type 1 (HIV-1) and malaria (10). Whether the addition of exogenous TLR agonists might further enhance the immunogenicity of viral vectors, however, remains unclear. The few studies that have explored the utility of TLR adjuvants with viral vectors have typically shown no or mild enhancement of antibody and T lymphocyte responses (7, 26). We therefore sought to determine systematically whether TLR ligands can modulate cellular immune responses elicited by a recombinant adenovirus serotype 26 (rAd26) vector in mice.C57BL/6 mice (n = 7 to 8/group) were immunized with a single injection of 3 × 10⁸ viral particles (vp) rAd26-Gag alone or combined with various TLR ligands (1). Vectors were mixed with soluble TLR agonists 1 h prior to intramuscular (i.m.) injection into both quadriceps muscles. Cellular immune responses were assessed by D^b/AL11 tetramer binding assays (3, 6), gamma interferon (IFN-γ) enzyme-linked immunospot (ELISPOT) assays (6), and multiparameter intracellular cytokine staining (ICS) assays (14). As shown in Fig. ​Fig.11 A, immunization with rAd26-Gag plus either 20 μg Pam3CSK (TLR1/2 ligand) (25), 20 μg Pam2CSK (TLR2/6 ligand) (9, 20), 10 μg flagellin (TLR5 ligand) (5, 8), 100 μg CLO97 (TLR7 ligand) (41), or 40 μg CpG (TLR9 ligand) (40) (all obtained from InvivoGen, San Diego, CA) elicited AL11-specific tetramer-positive responses (3, 6) that were similar to those detected in the unadjuvanted groups.Open in a separate windowFIG. 1.Antigen-specific CD8⁺ T cell responses elicited by rAd26-Gag are modulated by soluble TLR ligands. (A) C57BL/6 mice (n = 7 to 8 mice/group) were immunized once with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag combined with the following TLR ligands: 20 μg synthetic triacylated lipoprotein (Pam3CSK; TLR1/2 ligand), 20 μg synthetic diacylated lipoprotein (Pam2CSK; TLR 2/6 ligand), 100 μg poly(I:C) (TLR3 ligand), 10 μg LPS (TLR4 ligand), 10 μg flagellin (TLR5 ligand), 100 μg CLO97 (TLR7 ligand), or 40 μg unmethylated CpG-oligodeoxynucleotides (CpG; TLR9 ligand). Gag-specific cellular immune responses were assayed by D^b/AL11 tetramer binding assays at multiple time points following injection. (B) At week 4 following immunization, functional immune responses from mice immunized with rAd26 vaccine alone or with 10 μg LPS or 100 μg poly(I:C) were assessed by IFN-γ ELISPOT assays in response to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13. (C) Assessment of the dose response of LPS (10 μg, 2 μg, 0.4 μg) and poly(I:C) (100 μg, 20 μg, 4 μg) with rAd26-Gag (n = 4 mice/group) by D^b/AL11 tetramer binding assays. (D) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group), and Gag-specific CD8⁺ T cell responses in splenocytes were assessed 4 weeks after vaccination by intracellular cytokine assays for IFN-γ, TNF-α, IL-2, and CD107. Responses to pooled Gag peptides are presented for each individual combination of functions and collated as the number of functions elaborated as a percent of total CD8⁺ T lymphocytes (insert; bar graph) and as the fraction of Gag-specific CD8⁺ T lymphocytes (insert; pie charts). Mean responses with standard errors are shown (*, P < 0.001; **, P < 0.05; two-tailed t test).The TLR3 ligand poly(I:C) (InvivoGen, San Diego, CA), however, markedly suppressed responses to the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). This finding contrasts with prior reports demonstrating its adjuvanticity for protein antigen vaccines (22, 34, 37). By day 28, mice that received the vaccine plus 100 μg poly(I:C) developed Gag-specific CD8⁺ T lymphocyte responses that were significantly lower (1.7%) than those of mice that received the vaccine alone (5.4%; P < 0.001; two-tailed t test). Similarly, IFN-γ ELISPOT responses in mice that received poly(I:C) were lower than those observed in the unadjuvanted group (Fig. ​(Fig.1B)1B) (6). In a dose response study (Fig. ​(Fig.1C),1C), 100-μg, 20-μg, and 4-μg doses of poly(I:C) all resulted in diminished tetramer-positive responses.In contrast, the TLR4 ligand lipopolysaccharide (LPS) (Ultrapure LPS from Escherichia coli 0111:B4; InvivoGen, San Diego, CA) substantially enhanced Gag-specific CD8⁺ T lymphocyte responses elicited by the rAd26-Gag vaccine (Fig. ​(Fig.1A).1A). At day 28, tetramer-positive responses in mice that received the vaccine plus 10 μg LPS (9.6%) were significantly higher than those in the unadjuvanted group (5.4%; P = 0.04). Moreover, IFN-γ ELISPOT responses (6, 21) to pooled Gag peptides, the CD8⁺ T lymphocyte epitopes AL11 and KV9, and the CD4⁺ T lymphocyte epitope DD13 were greater in mice that received the vaccine with LPS than in mice that received the vaccine alone at week 4 after immunization (P = 0.02) (Fig. ​(Fig.1B).1B). To further quantify this effect, mice were immunized once i.m. (n = 4 mice/group) with rAd26-Gag with various doses of LPS (10 μg, 2 μg, 0.4 μg). Tetramer-positive responses were enhanced by 10 μg and 2 μg LPS but not by 0.4 μg LPS (Fig. ​(Fig.1C),1C), indicating that this LPS effect was dose dependent. No overt clinical toxicities were observed by using these doses of LPS in mice.We next evaluated the functionality of CD8⁺ T lymphocyte responses by multiparameter ICS assays that assessed IFN-γ, tumor necrosis factor alpha (TNF-α), interleukin-2 (IL-2), and the cytotoxic degranulation marker CD107 expression at week 4 following immunization with rAd26-Gag alone, rAd26-Gag with 2 μg LPS, or rAd26-Gag with 20 μg poly(I:C) (n = 4 to 8 mice/group) (15). As shown in Fig. ​Fig.1D,1D, the addition of LPS significantly enhanced not only the overall magnitude of Gag-specific CD8⁺ T lymphocyte responses (P = 0.04) but also the fraction of Gag-specific CD8⁺ T lymphocytes that expressed two or more effector functions (P = 0.04). In particular, the LPS-adjuvanted group induced higher levels of single-function CD107⁺, 2-function TNF-α⁺ CD107⁺, as well as 3-function IFN-γ⁺ TNF-α⁺ CD107⁺ CD8⁺ T lymphocytes than mice that received rAd26-Gag alone. These data show that LPS enhanced both the magnitude and functionality of antigen-specific cellular responses elicited by rAd26-Gag. In contrast, the addition of poly(I:C) diminished both the overall magnitude of Gag-specific responses and the fraction of these responses that were multifunctional.We further characterized the opposing effects of poly(I:C) and LPS by administering the rAd26-Gag vaccine with both poly(I:C) and LPS. C57BL/6 mice (n = 4 mice/group) were immunized with a single injection of rAd26-Gag alone or with 10 μg LPS, 60 μg poly(I:C), or both TLR ligands. As shown in Fig. ​Fig.22 A, administration of both TLR ligands resulted in reduced Gag-specific responses, suggesting that the suppressive effect of poly(I:C) was dominant over the enhancing effect of LPS. To determine the durability of the effects of poly(I:C) and LPS, C57BL/6 mice were primed with rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) (n = 4 mice/group) and were boosted on day 35 with a single i.m. injection of the heterologous vector rAd5HVR48(1-7) also expressing simian immunodeficiency virus (SIV) Gag (32). As shown in Fig. ​Fig.2B,2B, the mice that received poly(I:C) with the priming immunization responded to the boosting immunization with Gag-specific responses that were comparable to those observed in the mice that received rAd26-Gag alone. In contrast, mice that received LPS with the priming immunization exhibited sustained enhanced Gag-specific tetramer and ELISPOT responses, demonstrating the proliferative potential of antigen-specific CD8⁺ T lymphocytes elicited by the LPS-adjuvanted rAd26-Gag vaccine.Open in a separate windowFIG. 2.Dominant suppressive effect of poly(I:C) over LPS with the rAd26-Gag vaccine. (A) Mice were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 20 μg poly(I:C), 2 μg LPS, or both poly(I:C) and LPS (n = 4 mice/group). Gag-specific CD8⁺ T lymphocyte responses were assessed by D^b/AL11 tetramer binding assays and IFN-γ ELISPOT assays 4 weeks after immunization. (B) Mice were primed once with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C) and then boosted (↓) with 3 × 10⁸ vp rAd5HVR48(1-7) at week 5. Gag-specific cellular immune responses were assessed by D^b/AL11 tetramer binding assays and by IFN-γ ELISPOT responses at week 4 postboost. Mean responses with standard errors are shown.We next investigated whether the mechanism underlying the immunomodulatory effects of LPS and poly(I:C) involved the expected TLR signaling pathways. Although LPS and poly(I:C) are chiefly considered TLR ligands, poly(I:C) can also signal through the intracellular sensor MDA-5 (14), and both LPS and poly(I:C) may activate inflammasomes through Nalp3 (12, 28). To explore whether the effects of LPS and poly(I:C) involved TLR signaling, we utilized C57BL/6 mice lacking TRIF (Jackson Laboratory, Bar Harbor, ME), which is utilized by TLR3, or C57BL/6 mice lacking MyD88 (provided by S. Akira and B. Pulendran), which is utilized by the majority of TLRs. In particular, TLR4 signals through both TRIF and MyD88. Wild-type, MyD88^−/−, and TRIF^−/− mice (n = 4 mice/group) were immunized with rAd26-Gag vaccine alone or with 2 μg LPS or 20 μg poly(I:C). As shown in Fig. ​Fig.3,3, the adjuvant activity of LPS was abrogated in both MyD88^−/− and TRIF^−/− mice (Fig. 3A and B), suggesting that the adjuvanticity of the TLR4 ligand LPS was dependent on both MyD88 and TRIF, as expected. In contrast, the suppressive effect of poly(I:C) was observed in MyD88^−/− mice but not in TRIF^−/− mice (Fig. 3A and B), indicating that the suppressive effect of the TLR3 ligand poly(I:C) was dependent on TRIF, rather than MDA-5 or nonspecific effects (14, 39). These data confirm that the immunomodulatory effects of LPS and poly(I:C) were dependent on the expected TLR signaling pathways.Open in a separate windowFIG. 3.The immunomodulatory effects of poly(I:C) and LPS are TLR dependent. MyD88−/− and TRIF−/− mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁸ vp rAd26-Gag alone or with 2 μg LPS or 20 μg poly(I:C). (A) D^b/AL11 tetramer binding assays were performed at multiple time points following injection, and (B) IFN-γ ELISPOT responses were assessed 4 weeks after immunization. Mean responses with standard errors are shown.LPS is not a likely adjuvant for clinical development as a result of its toxicities, and alternative TLR4 ligands have been developed for potential clinical use. In particular, monophosphoryl lipid A (MPLA) is an LPS derivative that retains the immunologically active lipid A portion of the parent molecule (23, 27). The reduced toxicity of MPLA is attributed to the preferential recruitment of TRIF upon TLR4 activation, resulting in decreased induction of inflammatory cytokines (18). To determine if MPLA can similarly adjuvant cellular immune responses elicited by rAd26-Gag, C57BL/6 mice were immunized with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag alone or with 5 μg MPLA (derived from Salmonella enterica serovar Minnesota R595 LPS; InvivoGen, San Diego, CA) (n = 4 mice/group). This optimal dose of MPLA was selected by dose response studies (data not shown). As shown in Fig. ​Fig.44 A, Gag-specific IFN-γ ELISPOT responses to the lowest dose of vector were essentially undetectable in the unadjuvanted group, consistent with prior observations (1). In contrast, clear responses were observed in the mice that received 3 × 10⁷ vp rAd26-Gag with MPLA (P < 0.01; two-tailed t test). Mice that received the 3 × 10⁸ vp and 3 × 10⁹ vp doses of rAd26-Gag with MPLA also exhibited higher Gag-specific cellular immune responses than the unadjuvanted groups (P < 0.01). Functionality of these Gag-specific CD8⁺ T lymphocyte responses, as measured by multiparameter ICS assays assessing IFN-γ, TNF-α, IL-2, and CD107 expression, was also greater in mice that received rAd26-Gag with MPLA compared with rAd26-Gag (P < 0.05 for the lowest dose group) (Fig. ​(Fig.4B).4B). Thus, the TLR4 ligand MPLA also augmented antigen-specific CD8⁺ T lymphocyte responses elicited by rAd26-Gag.Open in a separate windowFIG. 4.The TLR4 ligand MPLA augments the immunogenicity of rAd26-Gag. C57BL/6 mice (n = 4 mice/group) were immunized once i.m. with 3 × 10⁷, 3 × 10⁸, or 3 × 10⁹ vp rAd26-Gag with or without 5 μg MPLA. Gag-specific cellular immune responses were assessed 4 weeks after immunization by IFN-γ ELISPOT responses (*, P < 0.01 for responses to pooled Gag peptides; two-tailed t test) (A) and by ICS for IFN-γ, TNF-α, IL-2, and CD107 (B). Responses to pooled Gag peptides in mice immunized with 3 × 10⁷ vp rAd26-Gag with or without 5 μg MPLA are presented for each individual combination of functions and collated as the number of functions as a fraction of the total Gag-specific CD8⁺ T lymphocyte response (insert; pie charts) (**, P < 0.05). (C) Cytokine levels were measured in sera of mice 8 h after immunization with 3 × 10⁸ vp rAd26-Gag alone or 3 × 10⁸ vp rAd26-Gag with 5 μg MPLA or 2 μg LPS (n = 4 mice/group). Mean responses with standard errors are shown.To explore differences in acute inflammatory responses following MPLA and LPS administration, serum levels of IL-1α, IL-6, granulocyte colony-stimulating factor (G-CSF), and IP-10 were assessed 8 h after vaccination in duplicate using multiplexed fluorescent bead-based immunoassays (Millipore, Billerica, MA) and analyzed on the Luminex 100 IS (Luminex, Austin, TX). As shown in Fig. ​Fig.4C,4C, mice that received MPLA had lower levels of the MyD88-associated acute proinflammatory cytokines IL-1α and IL-6 than mice that received LPS, as expected. Levels of IP-10 and G-CSF, which are associated with TRIF activation (18), were comparable (Fig. ​(Fig.4B).4B). These data confirm that MPLA resulted in lower levels of systemic inflammatory cytokine secretion than LPS.Optimization of the immunogenicity of viral vectors is an important research priority. However, there have been few reports addressing the potential use of adjuvants together with viral vectors. Combining alum with rAd35 elicited improved antibody responses to a malaria antigen (24), and the addition of TLR9 agonists (CpGs) resulted in paradoxically diminished immune responses elicited by a rAd5 vector but improved protection against a cancer antigen (13). Most recently, Appledorn et al. reported enhanced antigen-specific T lymphocyte responses with the coadministration of a rAd vector engineered to express a novel TLR5 agonist (4). Our study extends these findings and represents the first systematic investigation of the capacity of a panel of soluble TLR ligands to modulate rAd-elicited CD8⁺ T lymphocyte responses.The TLR agonists that modulated vaccine-elicited immune responses in this study included poly(I:C), LPS, and MPLA. These ligands have all been reported to augment CD8⁺ T lymphocyte responses elicited by peptide or protein vaccines (11, 22, 31, 33, 42), presumably through enhanced cross-presentation (34, 35). TLR signaling has been shown to be important for virus-elicited CD8⁺ T lymphocyte responses (38), often through activation of multiple TLRs or other pattern recognition receptors (30). The activation of TLR4 by LPS or MPLA with a viral vector most likely provides an additive or synergistic signal, probably resulting in enhanced APC maturation in the appropriate cytokine milieu. Moreover, immunization of the viral vector and LPS at different sites abrogated the observed adjuvanticity (data not shown), indicating that TLR4 adjuvanticity involves a local mechanism of action. However, the mechanism by which a TLR3 agonist suppresses immunogenicity of a viral vector remains unclear. It is possible that the high levels of type I interferon elicited by poly(I:C) (data not shown) may limit expression from the rAd26 vector. Alternatively, poly(I:C) has been reported to elicit IL-10 secretion, and this suppressive cytokine may limit CD8⁺ T cell proliferation (22, 36). The unexpected suppressive activity of poly(I:C) illustrates the inherent complexity of viral vectors compared to protein-based vaccines (16, 37).Our data demonstrate that antigen-specific CD8⁺ T lymphocyte responses elicited by a rAd26-Gag vaccine vector can be both positively and negatively modulated by soluble TLR ligands, and the mechanism underlying these observations involves the expected TRIF and MyD88 signaling pathways. In particular, the TLR4 ligands LPS and MPLA substantially augmented the magnitude and functionality of antigen-specific cellular immune responses elicited by this vaccine vector. These findings suggest that TLR ligands, particularly MPLA, deserve further exploration as potential adjuvants to improve the immunogenicity and protective efficacy of viral vaccine vectors.     

The Immunodominance Change and Protection of CD4+ T-Cell Responses Elicited by an Envelope Protein Domain III-Based Tetravalent Dengue Vaccine in Mice

Dengue is the leading cause of mosquito-borne viral infections and no vaccine is available now. Envelope protein domain III (ED3) is the major target for the binding of dengue virus neutralizing antibodies; however, the ED3-specifc T-cell response is less well understood. To investigate the T-cell responses to four serotypes of dengue virus (DENV-1 to 4), we immunized mice using either a tetravalent ED3-based DNA or protein vaccine, or combined both as a DNA prime-protein boost strategy (prime-boost). A significant serotype-dependent IFN-γ or IL-4 response was observed in mice immunized with either the DNA or protein vaccine. The IFN-γ response was dominant to DENV-1 to 3, whereas the IL-4 response was dominant to DENV-4. Although the similar IgG titers for the four serotypes were observed in mice immunized with the tetravalent vaccines, the neutralizing antibody titers varied and followed the order of 2 = 3>1>4. Interestingly, the lower IFN-γ response to DENV-4 is attributable to the immunodominance change between two CD4⁺ T-cell epitopes; one T-cell epitope located at E_349-363 of DENV-1 to 3 was more immunogenic than the DENV-4 epitope E_313-327. Despite DENV-4 specific IFN-γ responses were suppressed by immunodominance change, either DENV-4-specific IFN-γ or neutralizing antibody responses were still recalled after DENV-4 challenge and contributed to virus clearance. Immunization with the prime-boost elicited both IFN-γ and neutralizing antibody responses and provided better protection than either DNA or protein immunization. Our findings shed light on how ED3-based tetravalent dengue vaccines sharpen host CD4 T-cell responses and contribute to protection against dengue virus.     

HIV-1 Conserved Elements p24CE DNA Vaccine Induces Humoral Immune Responses with Broad Epitope Recognition in Macaques

To target immune responses towards invariable regions of the virus, we engineered DNA-based immunogens encoding conserved elements (CE) of HIV-1 p24^gag. This conserved element vaccine is designed to avoid decoy epitopes by focusing responses to critical viral elements. We previously reported that vaccination of macaques with p24CE DNA induced robust cellular immune responses to CE that were not elicited upon wild type p55^gag DNA vaccination. p24CE DNA priming followed by p55^gag DNA boost provided a novel strategy to increase the magnitude and breadth of the cellular immune responses to HIV-1 Gag, including the induction of strong, multifunctional T-cell responses targeting epitopes within CE. Here, we examined the humoral responses induced upon p24CE DNA or p55^gag DNA vaccination in macaques and found that although both vaccines induced robust p24^gag binding antibody responses, the responses induced by p24CE DNA showed a unique broad range of linear epitope recognition. In contrast, antibodies elicited by p55^gag DNA vaccine failed to recognize p24CE protein and did not recognize linear epitopes spanning the CE. Interestingly, boosting of p24CE DNA primed animals with p55^gag DNA resulted in augmentation of antibodies able to recognize p24^gag as well as the p24CE proteins, thereby inducing broadest immunity. Our results indicate that an effectively directed vaccine strategy that includes priming with the conserved element vaccine followed by boost with the complete immunogen induces broad cellular and humoral immunity focused on the conserved regions of the virus. This novel and effective strategy to broaden responses could be applied against other antigens of highly diverse pathogens.     

Increased Generation of HIV-1 gp120-Reactive CD8+ T Cells by a DNA Vaccine Construct Encoding the Chemokine CCL3

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV-1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8⁺ T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.     

HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted.     

Broad HIV-1 neutralization mediated by CD4-binding site antibodies

We have identified several patient sera showing potent and broad HIV-1 neutralization. Using antibody adsorption and elution from selected gp120 variants, the neutralizing specificities of the two most broadly reactive sera were mapped to the primary receptor CD4-binding region of HIV-1 gp120. Novel antibodies to the CD4-binding site are elicited in some HIV-1-infected individuals, and new approaches to present this conserved region of gp120 to the immune system may result in improved vaccine immunogens.     

Protective Antibody and CD8+ T-Cell Responses to the Plasmodium falciparum Circumsporozoite Protein Induced by a Nanoparticle Vaccine

Background

The worldwide burden of malaria remains a major public health problem due, in part, to the lack of an effective vaccine against the Plasmodium falciparum parasite. An effective vaccine will most likely require the induction of antigen specific CD8⁺ and CD4⁺ T-cells as well as long-lasting antibody responses all working in concert to eliminate the infection. We report here the effective modification of a self-assembling protein nanoparticle (SAPN) vaccine previously proven effective in control of a P. berghei infection in a rodent model to now present B- and T-cell epitopes of the human malaria parasite P. falciparum in a platform capable of being used in human subjects.

Methodology/Principal Findings

To establish the basis for a SAPN-based vaccine, B- and CD8⁺ T-cell epitopes from the P. falciparum circumsporozoite protein (PfCSP) and the universal CD4 T-helper epitope PADRE were engineered into a versatile small protein (∼125 amino acids) that self-assembles into a spherical nanoparticle repetitively displaying the selected epitopes. P. falciparum epitope specific immune responses were evaluated in mice using a transgenic P. berghei malaria parasite of mice expressing the human malaria full-length P. falciparum circumsporozoite protein (Tg-Pb/PfCSP). We show that SAPN constructs, delivered in saline, can induce high-titer, long-lasting (1 year) protective antibody and poly-functional (IFNγ⁺, IL-2⁺) long-lived central memory CD8⁺ T-cells. Furthermore, we demonstrated that these Ab or CD8⁺ T–cells can independently provide sterile protection against a lethal challenge of the transgenic parasites.

Conclusion

The SAPN construct induces long-lasting antibody and cellular immune responses to epitope specific sequences of the P. falciparum circumsporozoite protein (PfCSP) and prevents infection in mice by a transgenic P. berghei parasite displaying the full length PfCSP.     

Autologous HIV-1 Clade-B Nef Peptides Elicit Increased Frequency,Breadth and Function of CD8+ T-Cells Compared to Consensus Peptides

Objective

To determine the function and phenotype of CD8⁺ T-cells targeting consensus and autologous sequences of entire HIV-1 Nef protein.

Methods

Multiparameter flow cytometry-based analysis was used to evaluate the responses of two treatment naïve HIV-infected individuals, during primary and the chronic phases of infection.

Results

A greater breadth and magnitude of CD8 IFN-γ responses to autologous compared to clade-B consensus peptides was observed in both subjects. Cross recognition between autologous and consensus peptides decreased in both subjects during progression from primary to chronic infection. The frequencies of TEMRA and TEM CD8⁺ T-cells targeting autologous peptides were higher than those targeting consensus peptides and were more polyfunctional (IFN-γ⁺ Gr-B⁺ CD107a⁺).

Conclusions

Our data indicate superior sensitivity and specificity of autologous peptides. The functional and maturational aspects of “real” versus “cross-recognized” responses were also found to differ, highlighting the importance of a sequence-specific approach towards understanding HIV immune response.     

IL—18DNA免疫对HIV—1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应。酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01)。IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用。     

IL-18 DNA免疫对HIV-1核酸疫苗诱导的免疫应答的影响

为了研究白细胞介素-18(IL-18)基因对人免疫缺陷病毒(HIV-1)核酸疫苗诱导免疫应答的影响,将人IL-18基因插入到真核表达载体pVAX1中,构建了真核表达载体pVAX1-IL-18;将pCI-neoGAG联合pVAX1-IL-18或者pCI-neoGAG单独免疫Balb/c小鼠,检测免疫小鼠的特异性抗体和IFN-γ,同时观察免疫小鼠脾淋巴细胞增殖和小鼠特异性细胞毒性T淋巴细胞(CTL)反应.酶切及测序结果表明成功地构建了人IL-18基因真核表达载体;与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的抗HIV-1p24抗体滴度降低(P<0.01);而与pCI-neoGAG免疫组比较,pCI-neoGAG联合pVAX1-IL-18免疫组小鼠血清的IFN-γ升高(P<0.01);pCI-neoGAG联合pVAX1-IL-18免疫组小鼠的脾淋巴细胞增殖实验刺激指数(SI)以及特异性CTL活性均高于pCI-neoGAG免疫组(P<0.01).IL-18基因联合HIV-1核酸疫苗免疫小鼠,可能增强特异性Th1细胞和CTL反应,白细胞介素-18基因对体液免疫有抑制作用.     

Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24^gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55^gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist.     

Autophagy and CD4+ T lymphocyte destruction by HIV-1

The first step of HIV-1 infection is mediated by the binding of envelope glycoproteins (Env) to CD4 and two major coreceptors, CCR5 or CXCR4. The HIV-1 strains that use CCR5 are involved in primo-infection whereas those HIV-1 strains that use CXCR4 play a major role in the demise of CD4+ T lymphocytes and a rapid progression toward AIDS. Notably, binding of X4 Env expressed on cells to CXCR4 triggers apoptosis of uninfected CD4+ T cells. We now have just demonstrated that, independently of HIV-1 replication, transfected or HIV-1-infected cells that express X4 Env induce autophagy and accumulation of Beclin 1 in uninfected CD4+ T lymphocytes via CXCR4. Moreover, autophagy is a prerequisite to Env-induced apoptosis in uninfected bystander T cells, and CD4+ T cells still undergo an Env-mediated cell death with autophagic features when apoptosis is inhibited. To the best of our knowledge, these findings represent the first example of autophagy triggered through binding of virus envelope proteins to a cellular receptor, without viral replication, leading to apoptosis. Here, we proposed hypotheses about the significance of Env-induced Beclin 1 accumulation in CD4+ T cell death and about the role of autophagy in HIV-1 infected cells depending on the coreceptor involved.     

Influence of Novel CD4 Binding-Defective HIV-1 Envelope Glycoprotein Immunogens on Neutralizing Antibody and T-Cell Responses in Nonhuman Primates

The high-affinity in vivo interaction between soluble HIV-1 envelope glycoprotein (Env) immunogens and primate CD4 results in conformational changes that alter the immunogenicity of the gp120 subunit. Because the conserved binding site on gp120 that directly interacts with CD4 is a major vaccine target, we sought to better understand the impact of in vivo Env-CD4 interactions during vaccination. Rhesus macaques were immunized with soluble wild-type (WT) Env trimers, and two trimer immunogens rendered CD4 binding defective through distinct mechanisms. In one variant, we introduced a mutation that directly disrupts CD4 binding (368D/R). In the second variant, we introduced three mutations (423I/M, 425N/K, and 431G/E) that disrupt CD4 binding indirectly by altering a gp120 subdomain known as the bridging sheet, which is required for locking Env into a stable interaction with CD4. Following immunization, Env-specific binding antibody titers and frequencies of Env-specific memory B cells were comparable between the groups. However, the quality of neutralizing antibody responses induced by the variants was distinctly different. Antibodies against the coreceptor binding site were elicited by WT trimers but not the CD4 binding-defective trimers, while antibodies against the CD4 binding site were elicited by the WT and the 423I/M, 425N/K, and 431G/E trimers but not the 368D/R trimers. Furthermore, the CD4 binding-defective trimer variants stimulated less potent neutralizing antibody activity against neutralization-sensitive viruses than WT trimers. Overall, our studies do not reveal any potential negative effects imparted by the in vivo interaction between WT Env and primate CD4 on the generation of functional T cells and antibodies in response to soluble Env vaccination.The HIV-1 Envs mediate the entry of the virus into target cells and are the only virally encoded proteins exposed on the surface of the virus. HIV-1 Env is the sole target for neutralizing antibodies (Abs) and therefore is an important component of a vaccine designed to elicit protective antibody responses (4, 20). The viral spike is a trimer comprised of three heterodimers of the exterior envelope glycoprotein, gp120, noncovalently attached to the transmembrane protein, gp41. The gp120 subunit binds the primary receptor, CD4 (7), to form or to expose the gp120 coreceptor binding elements, which interact with the viral coreceptor, primarily CCR5 (1, 9, 12, 45). The highly conserved coreceptor binding site (CoRbs) overlaps the gp120 bridging sheet and also contains both proximal and distal elements of V3 (18, 32, 43, 45).In attempts to mimic the native trimeric structure of Env present on the virus, various forms of soluble Env trimers were designed (reviewed in reference 14). One design consists of cleavage-defective trimers derived from the primary R5 isolate YU2 that possess a heterologous trimerization motif derived from T4 bacteriophage fibritin (F; YU2 gp140-F) (3, 21, 34, 40, 50, 51). We recently demonstrated that the immunization of monkeys, but not rabbits, with gp140-F trimers resulted in the generation of Abs directed against the CoRbs of gp120 capable of cross-neutralizing HIV-2 (15). CoRbs-directed Abs (also referred to as CD4-induced, or CD4i, Abs) also were elicited in rabbits transgenic for human CD4 (15). Taken together, these data strongly suggest that Env interacts with high-affinity primate CD4 in vivo, resulting in the formation, or exposure, of a highly immunogenic gp120 determinant that overlaps the CoRbs. Early in infection, the frequency of HIV-1-infected individuals with significant antibody responses against the CoRbs is high (8, 33), and CoRbs-directed antibody responses are elicited abundantly in humans inoculated with Env-based immunogens (15). Collectively, these data suggest that the recognition of the HIV-1 CoRbs by naïve B cells is greatly increased when Env is presented in complex with high-affinity primate CD4, leading to a productive Ab response against this epitope (41). With rare exceptions, the majority of CoRbs-directed monoclonal antibodies (MAbs) do not neutralize HIV-1 primary viruses in vitro, bringing into question the utility of this region as a relevant neutralization target (23, 31, 47, 49). Strategies aimed to diminish vaccine-elicited B-cell responses to the CoRbs, and shift responses toward more accessible neutralization targets, represent one approach to improve the design of Env-based vaccine candidates. The selective manipulation of Env immunogens to decrease their CD4 binding capacity may reduce the elicitation of CoRbs-directed Abs and circumvent potential occlusion effects of the conserved CD4 binding site caused by CD4 itself.In addition to the potential effects of in vivo Env-CD4 interactions on the Ab repertoire elicited by Env-based immunogens, interactions between Env and CD4 also may have consequences on CD4⁺ T-cell responses. CD4 is an important costimulatory molecule expressed on several subsets of T cells and antigen-presenting cells, and interactions with Env were shown to alter the function of CD4-expressing T cells in a number of in vitro systems (13, 37, 44). The elimination of the Env-CD4 interaction in the context of vaccination may be beneficial to improve the elicitation of helper T-cell responses and effective neutralizing Ab responses. In vivo evaluation in subjects possessing high-affinity CD4 (i.e., rhesus macaques or humans) of CD4 binding-competent and CD4 binding-deficient Env immunogens so far have not been described.To address these questions, we designed Env trimer variants rendered CD4 binding defective through two distinct mechanisms. In the first variant, the interaction between CD4 and HIV-1 Env was directly disrupted by the introduction of a mutation (368D/R) in the CD4 binding loop of the gp120 outer domain (29). This alteration abolishes the initial binding of CD4 and most CD4 binding site (CD4bs)-directed MAbs (42) to variant forms of gp120 and would be expected to do the same in the soluble stable timer context. The aim of the second variant was to decrease the CD4 binding affinity while preserving the antigenicity of the CD4bs (48). This variant was generated in the soluble gp140-F trimers by the introduction of three point mutations, 423I/M, 425N/K, and 431G/E, in the β20 strand region of gp120. These mutations were suggested to favor a helix rather than the β20/21 antiparallel strands visible in the gp120 structure (23, 31, 47, 49). In the monomeric context, mutations in the β20 strand region of gp120 abolish binding by CoRbs-directed Abs, presumably because the bridging sheet cannot form (48). The introduction of the 423I/M, 425N/K, and 431G/E mutations in the trimer context therefore should disrupt the normally high-affinity gp120-CD4 interaction, while recognition by CD4bs Abs would not be affected. Indeed, a recent study provides a mechanistic basis for the impact of these mutations on CD4 binding (52). This study revealed that CD4 interacts with gp120 by a two-step binding mechanism in which the first step involves a direct, but low-affinity, CD4 interaction with the gp120 outer domain, while the second step requires a conformational change in gp120 to fully stabilize the high-affinity gp120-CD4 interaction.Here, we exploit this two-step model to generate novel CD4 binding-defective soluble trimers that, unlike the 368D/R trimers, possess a CD4bs surface that retains recognition by well-described CD4bs Abs. By immunizing rhesus macaques with the wild-type (WT) and CD4 binding-defective trimer variants, we demonstrate that similar levels of Env-specific Ab and T-cell responses were elicited in the three groups, suggesting that in vivo interactions between CD4 binding-competent (WT) Env and CD4 do not measurably affect T-cell responses against Env in this immunization regimen. However, the quality of the Ab response was markedly different between the groups. As hypothesized, CoRbs-directed Abs were elicited only in animals inoculated with WT trimers and not in those inoculated with 368D/R or 423I/M, 425N/K, and 431G/E trimers (hereafter referred to as 368 and 423/425/431 trimers, respectively). Importantly, we show that the 423/425/431 trimers retain the capacity to elicit binding and neutralizing CD4bs-directed Abs. In conclusion, the results generated in this study suggest that CD4 engagement by the WT soluble Env trimers did not impair the overall magnitude of the elicited Env-specific antibody or T-cell responses. Furthermore, our data provide new insights into the characteristics of Env that impact immunogenicity. The data also provide a potential path forward for the design of Env immunogens that have the capacity to elicit neutralizing Abs against the conserved gp120 CD4 binding surface while eliminating both the elicitation of nonneutralizing CoRbs-directed Abs and the potential occlusion of the CD4 binding surface of gp120 by the engagement of the primary virus receptor, CD4.     

DNA Vaccine Delivered by a Needle-Free Injection Device Improves Potency of Priming for Antibody and CD8+ T-Cell Responses after rAd5 Boost in a Randomized Clinical Trial

Background

DNA vaccine immunogenicity has been limited by inefficient delivery. Needle-free delivery of DNA using a CO₂-powered Biojector® device was compared to delivery by needle and syringe and evaluated for safety and immunogenicity.

Methods

Forty adults, 18–50 years, were randomly assigned to intramuscular (IM) vaccinations with DNA vaccine, VRC-HIVDNA016-00-VP, (weeks 0, 4, 8) by Biojector® 2000™ or needle and syringe (N/S) and boosted IM at week 24 with VRC-HIVADV014-00-VP (rAd5) with N/S at 10¹⁰ or 10¹¹ particle units (PU). Equal numbers per assigned schedule had low (≤500) or high (>500) reciprocal titers of preexisting Ad5 neutralizing antibody.

Results

120 DNA and 39 rAd5 injections were given; 36 subjects completed follow-up research sample collections. IFN-γ ELISpot response rates were 17/19 (89%) for Biojector® and 13/17 (76%) for N/S delivery at Week 28 (4 weeks post rAd5 boost). The magnitude of ELISpot response was about 3-fold higher in Biojector® compared to N/S groups. Similar effects on response rates and magnitude were observed for CD8+, but not CD4+ T-cell responses by ICS. Env-specific antibody responses were about 10-fold higher in Biojector-primed subjects.

Conclusions

DNA vaccination by Biojector® was well-tolerated and compared to needle injection, primed for greater IFN-γ ELISpot, CD8+ T-cell, and antibody responses after rAd5 boosting.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00109629","term_id":"NCT00109629"}}NCT00109629