Identification of HIV gp41-specific antibodies that mediate killing of infected cells.

Antibodies that mediate killing of HIV-infected cells through antibody-dependent cellular cytotoxicity (ADCC) have been implicated in protection from HIV infection and disease progression. Despite these observations, these types of HIV antibodies are understudied compared to neutralizing antibodies. Here we describe four monoclonal antibodies (mAbs) obtained from one individual that target the HIV transmembrane protein, gp41, and mediate ADCC activity. These four mAbs arose from independent B cell lineages suggesting that in this individual, multiple B cell responses were induced by the gp41 antigen. Competition and phage peptide display mapping experiments suggested that two of the mAbs target epitopes in the cysteine loop that are highly conserved and a common target of HIV gp41-specific antibodies. The amino acid sequences that bind these mAbs are overlapping but distinct. The two other mAbs were competed by mAbs that target the C-terminal heptad repeat (CHR) and the fusion peptide proximal region (FPPR) and appear to both target a similar unique conformational epitope. These gp41-specific mAbs mediated killing of infected cells that express high levels of Env due to either pre-treatment with interferon or deletion of vpu to increase levels of BST-2/Tetherin. They also mediate killing of target cells coated with various forms of the gp41 protein, including full-length gp41, gp41 ectodomain or a mimetic of the gp41 stump. Unlike many ADCC mAbs that target HIV gp120, these gp41-mAbs are not dependent on Env structural changes associated with membrane-bound CD4 interaction. Overall, the characterization of these four new mAbs that target gp41 and mediate ADCC provides evidence for diverse gp41 B cell lineages with overlapping but distinct epitopes within an individual. Such antibodies that can target various forms of envelope protein could represent a common response to a relatively conserved HIV epitope for a vaccine.

HIV Envelope Glycoform Heterogeneity and Localized Diversity Govern the Initiation and Maturation of a V2 Apex Broadly Neutralizing Antibody Lineage.

Understanding how broadly neutralizing antibodies (bnAbs) to HIV envelope (Env) develop during natural infection can help guide the rational design of an HIV vaccine. Here, we described a bnAb lineage targeting the Env V2 apex and the Ab-Env co-evolution that led to development of neutralization breadth. The lineage Abs bore an anionic heavy chain complementarity-determining region 3 (CDRH3) of 25 amino acids, among the shortest known for this class of Abs, and achieved breadth with only 10% nucleotide somatic hypermutation and no insertions or deletions. The data suggested a role for Env glycoform heterogeneity in the activation of the lineage germline B cell. Finally, we showed that localized diversity at key V2 epitope residues drove bnAb maturation toward breadth, mirroring the Env evolution pattern described for another donor who developed V2-apex targeting bnAbs. Overall, these findings suggest potential strategies for vaccine approaches based on germline-targeting and serial immunogen design.

[Follicular helper T cells and HIV - United for better and worse]. / Lymphocytes T folliculaires helper et VIH - Unis pour le meilleur et pour le pire.

Follicular helper T cells (Tfh) have been discovered in lymph nodes and, since then, are the focus of very intensive research to understand their origin, differentiation and functions. Tfh interact with B cells in the secondary lymphoid organs leading to B cell differentiation and maturation. Tfh are particularly studied in pathological contexts such as autoimmune diseases and infection by the human immunodeficiency virus (HIV). In the context of HIV infection, broadly neutralizing antibodies have been identified in a few patients. The generation of these broadly neutralizing antibodies requires a long and complex maturation of B cells in the secondary lymphoid organs. Characterizing Tfh functions and the relation with the quality of antibodies in HIV infection might help in designing novel immunotherapies and vaccination strategies to induce broadly neutralizing antibodies.

Synthetic multivalent V3 glycopeptides display enhanced recognition by glycan-dependent HIV-1 broadly neutralizing antibodies.

We describe here the synthesis of novel multivalent HIV V3 domain glycopeptides and their binding to broadly neutralizing antibodies PGT128 and 10-1074. Our binding data reveal a distinct mode of antigen recognition by the two antibodies and further suggest that multivalent glycopeptides could mimic the neutralizing epitopes more efficiently than the monomeric glycopeptide.

Influence of the Envelope gp120 Phe 43 Cavity on HIV-1 Sensitivity to Antibody-Dependent Cell-Mediated Cytotoxicity Responses.

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cellular-mediated cytotoxicity (ADCC). HIV-1 has evolved sophisticated mechanisms to avoid the exposure of Env ADCC epitopes by downregulating CD4 and by limiting the overall amount of Env on the cell surface. In HIV-1, substitution of large residues such as histidine or tryptophan for serine 375 (S375H/W) in the gp120 Phe 43 cavity, where Phe 43 of CD4 contacts gp120, results in the spontaneous sampling of an Env conformation closer to the CD4-bound state. While residue S375 is well conserved in the majority of group M HIV-1 isolates, CRF01_AE strains have a naturally occurring histidine at this position (H375). Interestingly, CRF01_AE is the predominant circulating strain in Thailand, where the RV144 trial took place. In this trial, which resulted in a modest degree of protection, ADCC responses were identified as being part of the correlate of protection. Here we investigate the influence of the Phe 43 cavity on ADCC responses. Filling this cavity with a histidine or tryptophan residue in Env with a natural serine residue at this position (S375H/W) increased the susceptibility of HIV-1-infected cells to ADCC. Conversely, the replacement of His 375 by a serine residue (H375S) within HIV-1 CRF01_AE decreased the efficiency of the ADCC response. Our results raise the intriguing possibility that the presence of His 375 in the circulating strain where the RV144 trial was held contributed to the observed vaccine efficacy.IMPORTANCE HIV-1-infected cells presenting Env in the CD4-bound conformation on their surface are preferentially targeted by ADCC mediated by HIV-positive (HIV+) sera. Here we show that the gp120 Phe 43 cavity modulates the propensity of Env to sample this conformation and therefore affects the susceptibility of infected cells to ADCC. CRF01_AE HIV-1 strains have an unusual Phe 43 cavity-filling His 375 residue, which increases the propensity of Env to sample the CD4-bound conformation, thereby increasing susceptibility to ADCC.

Complementary and synergistic activities of anti-V3, CD4bs and CD4i antibodies derived from a single individual can cover a wide range of HIV-1 strains.

Antibodies with modest neutralizing activity and narrow breadth are commonly elicited in HIV-1. Here, we evaluated the complementary and synergistic activities of a set of monoclonal antibodies (MAb) isolated from a single patient, directed to V3, CD4 binding site (CD4bs), and CD4 induced (CD4i) epitopes. Despite low somatic hypermutation percentages in the variable regions, these MAbs covered viral strains from subtypes B, C, A and CRF01_AE and transmitted/founder viruses in terms of binding, neutralizing and antibody-dependent cell-mediated cytotoxicity (ADCC) activities. In addition, a combination of the anti-V3 and CD4bs MAbs showed a synergistic effect over the neutralization of HIV-1JR-FL. A humoral response from a single patient covered a wide range of viruses by complementary and synergistic activities of antibodies with different specificities. Inducing a set of narrow neutralizing antibodies, easier to induce than the broadly neutralizing antibodies, could be a strategy for developing an effective vaccine against HIV-1.

[Anti-HIV antibodies: multiple antiviral activities]. / Les anticorps anti-VIH - de multiples activités antivirales.

Sexual transmission is currently the major route of HIV infection worldwide. Neutralizing antibodies (IgG) have demonstrated their role in the protection from experimental challenge in non-human primate's model. However, these types of antibodies display very specific characteristics and are extremely difficult to induce. Interestingly, antibodies devoid of neutralizing activity have demonstrated additional inhibitory mechanisms dependant of their binding to Fc receptors expressed on antigen presenting cells. These cells may play decisive role at early sexual transmission as they have been proposed to be the first HIV target at the mucosal site. Data from in vivo studies and recent findings following clinical assays demonstrated the importance of these Fc-mediated antibodies dependant mechanism in protection against HIV. Therefore new vaccination strategies including the induction of such type of activities, in addition to neutralizing antibodies, should be developed.

Impact of IgA constant domain on HIV-1 neutralizing function of monoclonal antibody F425A1g8.

With the majority of HIV infections resulting from mucosal transmission, induction of an effective mucosal immune response is thought to be pivotal in preventing transmission. HIV-specific IgA, but not IgG, has been detected in the genital tract, seminal fluid, urethral swabs, urine, and vaginal wash samples of HIV-negative sex workers and HIV-status discordant couples. Purified mucosal and plasma IgA from some individuals with highly exposed, persistently seronegative status can neutralize infection and present cross-clade neutralization activity, though present at low levels. We generated a CD4-induced human mAb, F425A1g8, and characterized the impact of its isotype variants on HIV neutralizing activity. The result showed that, in contrast to little neutralization by the F425A1g8 IgG1 in the absence of sCD4, the IgA1 variant of the Ab displayed significant independent neutralization activity against a range of HIV clade B isolates in the absence of sCD4. Studies of the neutralizing function of IgA isotypes, and the functional relationship between different antigenic epitopes and IgA Abs, may also suggest strategies for the intervention of virus transmission and spread within the mucosa of the host, as well as serve to inform the design of vaccine strategies that may be more effective at preventing mucosal transmission. This research clearly suggests that IgA isotype, because of its unique molecular structure, may play an important role in HIV neutralization.

HIV incidence determination in the United States: a multiassay approach.

BACKGROUND: Accurate testing algorithms are needed for estimating human immunodeficiency virus (HIV) incidence from cross-sectional surveys. METHODS: We developed a multiassay algorithm (MAA) for HIV incidence that includes the BED capture enzyme immunoassay (BED-CEIA), an antibody avidity assay, HIV load, and CD4(+) T-cell count. We analyzed 1782 samples from 709 individuals in the United States who had a known duration of HIV infection (range, 0 to >8 years). Logistic regression with cubic splines was used to compare the performance of the MAA to the BED-CEIA and to determine the window period of the MAA. We compared the annual incidence estimated with the MAA to the annual incidence based on HIV seroconversion in a longitudinal cohort. RESULTS: The MAA had a window period of 141 days (95% confidence interval [CI], 94-150) and a very low false-recent misclassification rate (only 0.4% of 1474 samples from subjects infected for >1 year were misclassified as indicative of recent infection). In a cohort study, annual incidence based on HIV seroconversion was 1.04% (95% CI, .70%-1.55%). The incidence estimate obtained using the MAA was essentially identical: 0.97% (95% CI, .51%-1.71%). CONCLUSIONS: The MAA is as sensitive for detecting recent HIV infection as the BED-CEIA and has a very low rate of false-recent misclassification. It provides a powerful tool for cross-sectional HIV incidence determination.

HIV vaccine: hopes and hurdles.

The AIDS vaccine development effort has already been facing various scientific and economic challenges. The fundamental challenge resides at the level of understanding the basic biology of HIV-1 infection and an effective antiviral immune response. There is a need to design immunogens that can elicit cross-clade neutralizing antibodies (NAbs) along with effective T-cell responses against a wide variety of primary HIV isolates. We must exploit the capabilities of the vaccine-elicited cytotoxic T cells and the NAb responses in controlling HIV-1 replication. A coordinated approach is required to understand the intricacies involved in the basic immune responses against HIV infection as well as the cross-clade effectiveness of an AIDS vaccine.

Identifying the early post-HIV antibody seroconversion period.

BACKGROUND: Identifying persons with recent human immunodeficiency virus (HIV) antibody seroconversion is useful for treatment, research, and prevention, but the sensitivity and specificity of tests for this purpose are uncertain. METHODS: We used longitudinal specimens panels from 155 persons identified prior to HIV seroconversion to assess antibody-based methods for classifying persons as within 30, 60, or 90 days of seroconversion, including 2 incidence assays, a less-sensitive (LS) enzyme immunoassay (EIA), and the BED assay. RESULTS: Sensitivity and specificity, respectively, for identifying persons within 30 days of seroconversion were: 34%-57% and 98%-100% for 2 standard EIAs (employing a signal-to-cutoff ≤4.0; ≥1.0 defines HIV positive), 84% and 73% for the LS-EIA (≤0.2 cutoff), 88% and 72% for the BED (≤0.2 cutoff), and 43%-58% and 98% (≤3 bands) for 2 Western blot (WB) assays. By area under the receiver operator curves, the best test for identifying persons within 30 days of seroconversion was the number of bands on the Bio-Rad WB (0.90); within 60 days, the LS-EIA and BED (both 0.85); and for persons within 90 days the BED (0.86). CONCLUSIONS: Standard EIAs, Western blots, and HIV incidence assays provide useful information for identifying persons 30 to 90 days after seroconversion.

Delayed maturation of antibody avidity but not seroconversion in rhesus macaques infected with simian HIV during oral pre-exposure prophylaxis.

BACKGROUND: Pre-exposure prophylaxis (PrEP) is a novel intervention strategy for the prevention HIV transmission. Because several clinical trials are at various stages of completion, it is important to understand the impact of PrEP treatment on the development of the immune response to HIV, particularly in individuals who exhibit breakthrough infections despite PrEP. METHODS: A model of HIV infection, using rhesus macaques and the simian/human immunodeficiency virus (SHIV), was used to evaluate the effects of PrEP on the evolution of the humoral immune response. Time to seroconversion, neutralizing and binding antibody levels, and antibody avidity were measured in 12 rhesus macaques infected during daily or intermittent PrEP with FTC (emtricitabine) or Truvada (FTC/tenofovir combination) and compared with 11 untreated, simian HIV-infected controls. RESULTS: Macaques that became infected while receiving PrEP exhibited significantly lower peak virus loads during acute infection as compared with untreated animals. Although the timing of seroconversion and SHIV binding and neutralizing antibody levels were not impacted by treatment, lower maturation rates of antibody avidity for anti-p27, gp120, gp160, and gp41 were observed. CONCLUSIONS: This study suggests that reduced virus loads associated with PrEP treatment have little impact on timing of seroconversion and neutralizing/binding antibody levels; however, maturation of antibody avidity was suppressed.

HIV-1 exposed uninfected men who have sex with men have increased levels of salivary CC-chemokines associated with sexual behavior.

OBJECTIVES: To determine whether soluble molecules with known anti-HIV-1 activity are increased in saliva of HIV-1 exposed uninfected individuals of discordant couples of men who have sex with men (MSM), and whether the levels of these molecules are associated with genetic polymorphisms, sexual behavior and/or HIV-1 neutralizing capacity. METHODS: Saliva and PBMC were collected from exposed uninfected individuals (n=25), and low-risk controls (n=22). Levels of CCL2, CCL3, CCL4, CCL5 and CCL11 were detected by Luminex, and SLPI, LL-37, alpha-defensins and IgA2 were detected by ELISA. Single nucleotide polymorphisms (SNPs) were investigated using mass spectrometry or PCR-sequencing. HIV-1 neutralizing activity was assessed using PBMCbased neutralization assays. Self-reported questionnaires described sexual behavior. RESULTS: Exposed uninfected individuals had significantly higher levels of salivary CCL2, CCL4, CCL5 and CCL11 as compared with controls although genetic polymorphisms within the corresponding regions were equally distributed. IgA2 was also increased in exposed uninfected individuals, whereas neither CCL3, SLPI, LL-37 nor alpha-defensins differed between exposed uninfected individuals and controls. The HIV-1 neutralizing capacity of saliva was associated with higher levels of CC-chemokines (but not SLPI, LL-37, alpha-defensins or IgA2) in both exposed uninfected individuals and controls. The increased levels of CC-chemokines were associated with a higher frequency of unprotected oral sex and/or additional casual sex partners. CONCLUSION: HIV-1 exposed uninfected MSM had higher levels of salivary CC-chemokines compared with controls, this finding associated with sexual behavior rather than with genetic polymorphisms. The increased levels of CC-chemokines associated with HIV-1 neutralizing capacity in saliva.

Rapid degranulation of NK cells following activation by HIV-specific antibodies.

Ab-dependent cellular cytotoxicity (ADCC) Abs stimulate NK cell effector functions and play a role in protecting from and controlling viral infections. We characterized ADCC Abs in a cross-sectional cohort of 80 HIV-infected subjects not on antiretroviral therapy. We analyzed ADCC response by killing fluorescently labeled target cells, as well as expression of IFN-gamma and the degranulation marker CD107a from activated NK cells as measured by a novel intracellular cytokine assay. HIV-specific ADCC directed toward Envelope proteins were present in the majority of 80 untreated HIV-infected individuals measured by killing function. Similarly, most subjects had HIV-specific Abs that mediated degranulation or cytokine expression by NK cells. Interestingly, there was a poor correlation between ADCC-mediated killing of fluorescently labeled whole Envelope protein-pulsed cell lines and Ab-mediated expression of IFN-gamma by NK cells. However, in contrast to healthy donor NK cells, autologous patient NK cells more effectively degranulated granzyme B in response to ADCC activation. Activation of NK cells in response to stimulation by HIV-specific Abs occurs at least as rapidly as activation of Gag-specific CTLs. Our studies highlight the complexity of ab-mediated NK cell activation in HIV infection, and suggest new avenues toward studying the utility of ADCC in controlling HIV infection.

Excretion of human immunodeficiency virus type 1 through polarized epithelium by immunoglobulin A.

Human immunodeficiency virus (HIV) is transmitted primarily sexually across mucosal surfaces. After infection, HIV propagates initially in the lamina propria below the polarized epithelium and causes extensive destruction of mucosal T cells. Immunoglobulin A (IgA) antibodies, produced in the lamina propria and then transcytosed across the mucosal epithelium into the lumen, can be the first line of immune defense against HIV. Here, we used IgA monoclonal antibodies against HIV envelope proteins to investigate the abilities of polarized primate and human epithelial cells to excrete HIV virions from the basolateral to the apical surface via polymeric Ig receptor (pIgR)-mediated binding and the internalization of HIV-IgA immune complexes. African green monkey kidney cells expressing pIgR demonstrated HIV excretion that was dependent on the IgA concentration and the exposure time. Matched IgG antibodies with the same variable regions as the IgA antibodies and IgA antibodies to non-HIV antigens had no HIV excretory function. A mixture of two IgA anti-bodies against gp120 and gp41 showed a synergistic increase in the level of HIV excreted. The capacity for HIV excretion correlated with the ability of IgA antibodies to bind HIV and of the resulting immune complexes to bind pIgR. Consistent with the epithelial transcytosis of HIV-IgA immune complexes, the colocalization of HIV proteins and HIV-specific IgA was detected intracellularly by confocal microscopy. Our results suggest the potential of IgA antibodies to excrete HIV from mucosal lamina propria, thereby decreasing the viral burden, access to susceptible cells, and the chronic activation of the immune system.

Catalytic antibodies to HIV: physiological role and potential clinical utility.

Immunoglobulins (Igs) in uninfected humans recognize residues 421-433 located in the B cell superantigenic site (SAg) of the HIV envelope protein gp120 and catalyze its hydrolysis by a serine protease-like mechanism. The catalytic activity is encoded by germline Ig variable (V) region genes, and is expressed at robust levels by IgMs and IgAs but poorly by IgGs. Mucosal IgAs are highly catalytic and neutralize HIV, suggesting that they constitute a first line of defense against HIV. Lupus patients produce the Igs at enhanced levels. Homology of the 421-433 region with an endogenous retroviral sequence and a bacterial protein may provide clues about the antigen driving anti-SAg synthesis in lupus patients and uninfected subjects. The potency and breadth of HIV neutralization revives hopes of clinical application of catalytic anti-421-433 Igs as immunotherapeutic and topical microbicide reagents. Adaptive improvement of anti-SAg catalytic Igs in HIV infected subjects is not customary. Further study of the properties of the naturally occurring anti-SAg catalytic Igs should provide valuable guidance in designing a prophylactic vaccine that amplifies protective catalytic immunity to HIV.

Complement and antibodies: a dangerous liaison in HIV infection?

Due to ongoing recombination and mutations, HIV permanently escapes from neutralizing antibody (nAb) responses of the host. By the masking of epitopes or shedding of gp120, HIV-1 further impedes an efficient neutralization by Abs. Therefore, nAbs responses of the host are chasing behind a rapidly evolving virus and mainly non-neutralizing antibodies (non-nAbs) are present in the host. At the same time, complement deposition on immune-complexed HIV may counteract the immune response by enhancing the infection. On the other hand, complement-mediated lysis is a putative effector mechanism to control viral replication. Here we review the complex interplay between complement, neutralizing and non-neutralizing Abs during HIV infection and discuss the contribution of Abs and complement in blocking versus enhancing the course of infection.

Recombinant gp120 vaccine-induced antibodies inhibit clinical strains of HIV-1 in the presence of Fc receptor-bearing effector cells and correlate inversely with HIV infection rate.

Nonneutralizing Abs may play a role in protecting animals and humans from lentiviral infections. We explored the Ab-dependent, cell-mediated virus inhibition (ADCVI) Ab response to recombinant gp120 (rgp120) vaccination in sera from 530 participants in the Vax 004 trial. Serum ADCVI activity was measured against a clinical R5 strain of HIV-1 using peripheral blood mononuclear effector cells from healthy donors. The level of vaccine-induced ADCVI activity correlated inversely with the rate of acquiring HIV infection following vaccination, such that for every 10% increase in ADCVI activity, there was a 6.3% decrease in the hazard rate of infection (p=0.019). Some vaccinated individuals also mounted an ADCVI response against two other clinical R5 strains of HIV-1. However, ADCVI activity correlated poorly with neutralizing or CD4-gp120-blocking Ab activity measured against laboratory strains. Finally, the degree to which the ADCVI Ab response predicted the rate of infection was influenced by polymorphisms at the FcgammaRIIa and FcgammaRIIIa gene loci. These data indicate that rgp120 vaccination can elicit Abs with antiviral activity against clinical strains of HIV-1. However, such activity requires the presence of FcR-bearing effector cells. Our results provide further evidence that ADCVI may play a role in preventing HIV infection.

Increased efficacy of HIV-1 neutralization by antibodies at low CCR5 surface concentration.

It has been observed that some antibodies, including the CD4-induced (CD4i) antibody IgG X5 and the gp41-specific antibody IgG 2F5, exhibit higher neutralizing activity in PBMC-based assays than in cell line based assays [J.M. Binley, T. Wrin, B. Korber, M.B. Zwick, M. Wang, C. Chappey, G. Stiegler, R. Kunert, S. Zolla-Pazner, H. Katinger, C.J. Petropoulos, D.R. Burton, Comprehensive cross-clade neutralization analysis of a panel of anti-human immunodeficiency virus type 1 monoclonal antibodies, J. Virol. 78 (2004) 13232-13252]. It has been hypothesized that the lower CCR5 concentration on the surface of the CD4 T lymphocytes compared to that on cell lines used for the neutralization assays could be a contributing factor to the observed differences in neutralizing activity. To test this hypothesis and to further elucidate the contribution of CCR5 concentration differences on antibody neutralizing activity, we used a panel of HeLa cell lines with well-defined and differential surface concentrations of CCR5 and CD4 in a pseudovirus-based assay. We observed that the CCR5 cell surface concentration but not the CD4 concentration had a significant effect on the inhibitory activity of X5 and several other CD4i antibodies including 17b and m9, as well as that of the gp41-specifc antibodies 2F5 and 4E10 but not on that of the CD4 binding site antibody (CD4bs), b12. The 50% inhibitory concentration (IC50) decreased up to two orders of magnitude in cell lines with low CCR5 concentration corresponding to that in CD4 T cells used in PBMC-based assays (about 10(3) per cell) compared to cell lines with high CCR5 concentration (about 10(4) or more). Our results suggest that the CCR5 cell surface concentration could be a contributing factor to the high neutralizing activities of some antibodies in PBMC-based-assays but other factors could also play an important role. These findings could have implications for development of vaccine immunogens based on the epitopes of X5 and other CD4i antibodies, for elucidation of the mechanisms of HIV-1 neutralization by antibodies, and for design of novel therapeutic approaches.

Increased level and longevity of protective immune responses induced by DNA vaccine expressing the HIV-1 Env glycoprotein when combined with IL-21 and IL-15 gene delivery.

We investigated the ability of a plasmid-derived IL-21 delivered alone or in combination with the IL-15 gene to regulate immune responses to the HIV-1 envelope (Env) glycoprotein induced by DNA vaccination. Mice were injected with the gp140DeltaCFI(HXB2/89.6) vector expressing a modified Env glycoprotein with C-terminal mutations intended to mimic a fusion intermediate, in which the most divergent region encoding the variable V1, V2, and V3 domains of CXCR4-tropic HxB2 virus was replaced with the dual-tropic 89.6 viral strain. Using a recombinant vaccinia virus expressing 89.6 Env glycoprotein (vBD3) in a mouse challenge model, we observed that IL-21 plasmid produced sustained resistance to viral transmission when injected 5 days after DNA vaccination. Moreover, IL-21 in a synergistic manner with IL-15 expression vector augmented the vaccine-induced recall responses to the vBD3 challenge compared with those elicited by immunization in the presence of either cytokine alone. The synergistic combination of IL-21 and IL-15 plasmids promoted expansion of CD8+CD127+ memory T cell pools specific for a subdominant HLA-A2-restricted Env(121-129) epitope (KLTPLCVTL). Our results also show that coimmunization with IL-21 and IL-15 plasmid combination resulted in enhanced CD8+ T cell function that was partially independent of CD4+ T cell help in mediating protection against vBD3 challenge. Furthermore, the use of IL-21 and IL-15 genes was able to increase Ab-dependent cellular cytotoxicity and complement-dependent lysis of Env-expressing target cells through augmentation of Env-specific IgG Ab levels. These data indicate that the plasmid-delivered IL-21 and IL-15 can increase the magnitude of the response to DNA vaccines.