SARS-CoV-2 DNA Vaccine INO-4800 Induces Durable Immune Responses Capable of Being Boosted in a Phase 1 Open-Label Trial.

BACKGROUND: Additional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines that are safe and effective as primary vaccines and boosters remain urgently needed to combat the coronavirus disease 2019 (COVID-19) pandemic. We describe safety and durability of immune responses following 2 primary doses and a homologous booster dose of an investigational DNA vaccine (INO-4800) targeting full-length spike antigen. METHODS: Three dosage strengths of INO-4800 (0.5 mg, 1.0 mg, and 2.0 mg) were evaluated in 120 age-stratified healthy adults. Intradermal injection of INO-4800 followed by electroporation at 0 and 4 weeks preceded an optional booster 6-10.5 months after the second dose. RESULTS: INO-4800 appeared well tolerated with no treatment-related serious adverse events. Most adverse events were mild and did not increase in frequency with age and subsequent dosing. A durable antibody response was observed 6 months following the second dose; a homologous booster dose significantly increased immune responses. Cytokine-producing T cells and activated CD8+ T cells with lytic potential were significantly increased in the 2.0-mg dose group. CONCLUSIONS: INO-4800 was well tolerated in a 2-dose primary series and homologous booster in all adults, including elderly participants. These results support further development of INO-4800 for use as primary vaccine and booster. CLINICAL TRIALS REGISTRATION: NCT04336410.

CD8+ T Cells Impact Rising PSA in Biochemically Relapsed Cancer Patients Using Immunotherapy Targeting Tumor-Associated Antigens.

The management of men with prostate cancer (PCa) with biochemical recurrence following local definitive therapy remains controversial. Early use of androgen deprivation therapy (ADT) leads to significant side effects. Developing an alternative, clinically effective, and well-tolerated therapy remains an unmet clinical need. INO-5150 is a synthetic DNA therapy that includes plasmids encoding for prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA), and INO-9012 is a synthetic DNA plasmid encoding for interleukin-12 (IL-12). This phase 1/2, open-label, multi-center study enrolled men with PCa with rising PSA after surgery and/or radiation therapy. Patients were enrolled into one of four treatment arms: arm A, 2 mg of INO-5150; arm B, 8.5 mg of INO-5150; arm C, 2 mg of INO-5150 + 1 mg of INO-9012; and arm D, 8.5 mg of INO-5150 + 1 mg of INO-9012. Patients received study drug with electroporation on day 0 and on weeks 3, 12, and 24, and they were followed for up to 72 weeks. Sixty-two patients were enrolled. Treatment was well tolerated. 81% (50/62) of patients completed all visits. 85% (53/62) remained progression-free at 72 weeks. PSA doubling time (PSADT) was increased when assessed in patients with day 0 PSADT ≤12 months. Immunogenicity was observed in 76% (47/62) of patients by multiple assessments. Analysis indicated that CD38 and perforin co-positive CD8 T cell frequency correlated with attenuated PSA rise (p = 0.05, n = 50).

Seroprotection of HIV-infected subjects after influenza A(H1N1) vaccination is directly associated with baseline frequency of naive T cells.

Human immunodeficiency virus type 1 (HIV-1)-infected individuals, despite receipt of antiretroviral therapy (ART), often have impaired vaccine responses. We examined the role that immune activation and cellular phenotypes play in influenza A(H1N1) vaccine responsiveness in HIV-infected subjects receiving ART. Subjects received the H1N1 vaccine (15-µg dose; Novartis), and antibody titers at baseline and after immunization were evaluated. Subjects were classified as responders if, by week 3, seroprotection guidelines were met. Responders had higher percentages of baseline naive T cells and lower percentages of terminally differentiated T cells, compared with nonresponders. Additionally, the naive CD4(+) T-cell percentage and age were negatively correlated. Preservation of naive T-cell populations by starting therapy early could impact vaccine responses against influenza virus and other pathogens, especially as this population ages.

Strong immunogenicity & protection in mice with PlaCCine: A COVID-19 DNA vaccine formulated with a functional polymer.

DNA- based vaccines have demonstrated the potential as a safe and effective modality. PlaCCine, a DNA-based vaccine approach described subsequently relies on a synthetic DNA delivery system and is independent of virus or device. The synthetic functionalized polymer combined with DNA demonstrated stability over 12 months at 4C and for one month at 25C. Transfection efficiency compared to naked DNA increased by 5-15-fold in murine skeletal muscle. Studies of DNA vaccines expressing spike proteins from variants D614G (pVAC15), Delta (pVAC16), or a D614G + Delta combination (pVAC17) were conducted. Mice immunized intramuscular injection (IM) with pVAC15, pVAC16 or pVAC17 formulated with functionalized polymer and adjuvant resulted in induction of spike-specific humoral and cellular responses. Antibody responses were observed after one immunization. And endpoint IgG titers increased to greater than 1x 105 two weeks after the second injection. Neutralizing antibodies as determined by a pseudovirus competition assay were observed following vaccination with pVAC15, pVAC16 or pVAC17. Spike specific T cell immune responses were also observed following vaccination and flow cytometry analysis demonstrated the cellular immune responses included both CD4 and CD8 spike specific T cells. The immune responses in vaccinated mice were maintained for up to 14 months after vaccination. In an immunization and challenge study of K18 hACE2 transgenic mice pVAC15, pVAC16 and pVAC17 induced immune responses lead to decreased lung viral loads by greater than 90 % along with improved clinical score. These findings suggest that PlaCCine DNA vaccines are effective and stable and further development against emerging SARS-CoV-2 variants is warranted.

Plasmodium inui infection reduces the efficacy of a simian immunodeficiency virus DNA vaccine in a rhesus macaque model through alteration of the vaccine-induced immune response.

Human immunodeficiency virus type 1 and malaria are co-endemic in many areas. We evaluated the effects of Plasmodium inui infection on the performance of a simian immunodeficiency virus (SIV) DNA vaccine. Rhesus macaques were infected with P. inui by transfusion of whole blood from a persistently infected animal. Animals with and animals without P. inui infection were then vaccinated 4 times with an SIV DNA vaccine encoding SIVgag, SIVpol, and SIVenv. Animals were subsequently challenged with thirty 50% rhesus monkey infectious doses of SIVmac251 6 weeks after the last vaccination. P. inui-infected immunized animals showed a significantly higher viral load than animals without P. inui infection (P = .010, by the Wilcoxon rank sum test). The higher viral loads in the P. inui-infected animals were durable and were observed at all sampling time points across the study (P = .00245, by the Wilcoxon rank test). The P. inui-infected animals also had correspondingly lower CD4(+) cell counts. There were fewer vaccine-specific CD4(+) and CD8(+) cells in the P. inui-infected animals, compared with uninfected animals. Of importance, P. inui infection seemed to decrease the number of CD8(+) cells that could proliferate or secrete interferon γ, although the number of CD8(+) cells capable of secreting tumor necrosis factor α following in vitro stimulation was increased. This study demonstrated that P. inui infection had an influence on the immune response to an SIV DNA vaccine and decreased the vaccine's efficacy.

DNA-delivered antibody cocktail exhibits improved pharmacokinetics and confers prophylactic protection against SARS-CoV-2.

Monoclonal antibody therapy has played an important role against SARS-CoV-2. Strategies to deliver functional, antibody-based therapeutics with improved in vivo durability are needed to supplement current efforts and reach underserved populations. Here, we compare recombinant mAbs COV2-2196 and COV2-2130, which compromise clinical cocktail Tixagevimab/Cilgavimab, with optimized nucleic acid-launched forms. Functional profiling of in vivo-expressed, DNA-encoded monoclonal antibodies (DMAbs) demonstrated similar specificity, broad antiviral potency and equivalent protective efficacy in multiple animal challenge models of SARS-CoV-2 prophylaxis compared to protein delivery. In PK studies, DNA-delivery drove significant serum antibody titers that were better maintained compared to protein administration. Furthermore, cryo-EM studies performed on serum-derived DMAbs provide the first high-resolution visualization of in vivo-launched antibodies, revealing new interactions that may promote cooperative binding to trimeric antigen and broad activity against VoC including Omicron lineages. These data support the further study of DMAb technology in the development and delivery of valuable biologics.

CD8+ cell depletion of SHIV89.6P-infected macaques induces CD4+ T cell proliferation that contributes to increased viral loads.

Previous studies have shown that depletion of CD8(+) cells during acute and chronic simian immunodeficiency virus (SIV) infection leads to increased viral replication, morbidity, and mortality, which have been attributed to loss of CD8(+) T cell-mediated control of SIV. However, these studies did not exclude that CD8(+) cell depletion increased homeostatic proliferation of CD4(+) T cells, resulting in increased viral targets and, therefore, viral rebound. Chronically SHIV89.6P-infected cynomolgus macaques were CD8(+) cell-depleted, and the frequency, cell number, and phenotype of CD4(+) T cells and viral infection were examined using flow cytometry and quantitative real-time PCR. The frequency and number of Ki-67-expressing CD4(+) T cells were increased with CD8(+) cell depletion. This proliferation of CD4(+) T cells occurred even in animals with no rebound of viral loads. Most of the proliferating cells were effector memory CD4(+) T cells. Plasma simian HIV (SHIV) RNA copies positively correlated with proliferating CD4(+) T cells and SHIV DNA copies in Ki-67(+) CD4(+) T cells. Although this study does not exclude an important role for virus-specific CD8(+) T cells in SIV and SHIV infection, our data suggest that homeostatic proliferation is an important contributor to increases in plasma viremia that follow CD8(+) cell depletion.

Phase 1 study of safety, tolerability and immunogenicity of the human telomerase (hTERT)-encoded DNA plasmids INO-1400 and INO-1401 with or without IL-12 DNA plasmid INO-9012 in adult patients with solid tumors.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is frequently classified as a 'universal' tumor associated antigen due to its expression in a vast number of cancers. We evaluated plasmid DNA-encoded hTERT as an immunotherapy across nine cancer types. METHODS: A phase 1 clinical trial was conducted in adult patients with no evidence of disease following definitive surgery and standard therapy, who were at high risk of relapse. Plasmid DNA encoding one of two hTERT variants (INO-1400 or INO-1401) with or without plasmid DNA encoding interleukin 12 (IL-12) (INO-9012) was delivered intramuscularly concurrent with the application of the CELLECTRA constant-current electroporation device 4 times across 12 weeks. Safety assessments and immune monitoring against native (germline, non-mutated, non-plasmid matched) hTERT antigen were performed. The largest cohort of patients enrolled had pancreatic cancer, allowing for additional targeted assessments for this tumor type. RESULTS: Of the 93 enrolled patients who received at least one dose, 88 had at least one adverse event; the majority were grade 1 or 2, related to injection site. At 18 months, 54.8% (51/93) patients were disease-free, with median disease-free survival (DFS) not reached by end of study. For patients with pancreatic cancer, the median DFS was 9 months, with 41.4% of these patients remaining disease-free at 18 months. hTERT immunotherapy induced a de novo cellular immune response or enhanced pre-existing cellular responses to native hTERT in 96% (88/92) of patients with various cancer types. Treatment with INO-1400/INO-1401±INO-9012 drove hTERT-specific IFN-γ production, generated hTERT-specific CD4+ and CD8+ T cells expressing the activation marker CD38, and induced hTERT-specific activated CD8 +CTLs as defined by cells expressing perforin and granzymes. The addition of plasmid IL-12 adjuvant elicited higher magnitudes of cellular responses including IFN-γ production, activated CD4+ and CD8+ T cells, and activated CD8+CTLs. In a subset analysis of pancreatic cancer patients, the presence of immunotherapy-induced activated CD8+ T cells expressing PD-1, granzymes and perforin correlated with survival. CONCLUSIONS: Plasmid DNA-encoded hTERT/IL-12 DNA immunotherapy was well-tolerated, immune responses were noted across all tumor types, and a specific CD8+ phenotype increased by the immunotherapy was significantly correlated with survival in patients with pancreatic cancer.

INO-4800 DNA vaccine induces neutralizing antibodies and T cell activity against global SARS-CoV-2 variants.

Global surveillance has identified emerging SARS-CoV-2 variants of concern (VOC) associated with broadened host specificity, pathogenicity, and immune evasion to vaccine-induced immunity. Here we compared humoral and cellular responses against SARS-CoV-2 VOC in subjects immunized with the DNA vaccine, INO-4800. INO-4800 vaccination induced neutralizing antibodies against all variants tested, with reduced levels detected against B.1.351. IFNγ T cell responses were fully maintained against all variants tested.

Safety and immunogenicity of INO-4800 DNA vaccine against SARS-CoV-2: A preliminary report of an open-label, Phase 1 clinical trial.

BACKGROUND: A vaccine against SARS-CoV-2 is of high urgency. Here the safety and immunogenicity induced by a DNA vaccine (INO-4800) targeting the full length spike antigen of SARS-CoV-2 are described. METHODS: INO-4800 was evaluated in two groups of 20 participants, receiving either 1.0 mg or 2.0 mg of vaccine intradermally followed by CELLECTRA® EP at 0 and 4 weeks. Thirty-nine subjects completed both doses; one subject in the 2.0 mg group discontinued trial participation prior to receiving the second dose. ClinicalTrials.gov identifier: NCT04336410. FINDINGS: The median age was 34.5, 55% (22/40) were men and 82.5% (33/40) white. Through week 8, only 6 related Grade 1 adverse events in 5 subjects were observed. None of these increased in frequency with the second administration. No serious adverse events were reported. All 38 subjects evaluable for immunogenicity had cellular and/or humoral immune responses following the second dose of INO-4800. By week 6, 95% (36/38) of the participants seroconverted based on their responses by generating binding (ELISA) and/or neutralizing antibodies (PRNT IC50), with responder geometric mean binding antibody titers of 655.5 [95% CI (255.6, 1681.0)] and 994.2 [95% CI (395.3, 2500.3)] in the 1.0 mg and 2.0 mg groups, respectively. For neutralizing antibody, 78% (14/18) and 84% (16/19) generated a response with corresponding geometric mean titers of 102.3 [95% CI (37.4, 280.3)] and 63.5 [95% CI (39.6, 101.8)], in the respective groups. By week 8, 74% (14/19) and 100% (19/19) of subjects generated T cell responses by IFN-É£ ELISpot assay with the median SFU per 106 PBMC of 46 [95% CI (21.1, 142.2)] and 71 [95% CI (32.2, 194.4)] in the 1.0 mg and 2.0 mg groups, respectively. Flow cytometry demonstrated a T cell response, dominated by CD8+ T cells co-producing IFN-É£ and TNF-α, without increase in IL-4. INTERPRETATION: INO-4800 demonstrated excellent safety and tolerability and was immunogenic in 100% (38/38) of the vaccinated subjects by eliciting either or both humoral or cellular immune responses. FUNDING: Coalition for Epidemic Preparedness Innovations (CEPI).

Protection against simian/human immunodeficiency virus (SHIV) 89.6P in macaques after coimmunization with SHIV antigen and IL-15 plasmid.

The cell-mediated immune profile induced by a recombinant DNA vaccine was assessed in the simian/HIV (SHIV) and macaque model. The vaccine strategy included coimmunization of a DNA-based vaccine alone or in combination with an optimized plasmid encoding macaque IL-15 (pmacIL-15). We observed strong induction of vaccine-specific IFN-gamma-producing CD8(+) and CD4(+) effector T cells in the vaccination groups. Animals were subsequently challenged with 89.6p. The vaccine groups were protected from ongoing infection, and the IL-15 covaccinated group showed a more rapidly controlled infection than the group treated with DNA vaccine alone. Lymphocytes isolated from the group covaccinated with pmacIL-15 had higher cellular proliferative responses than lymphocytes isolated from the macaques that received SHIV DNA alone. Vaccine antigen activation of lymphocytes was also studied for a series of immunological molecules. Although mRNA for IFN-gamma was up-regulated after antigen stimulation, the inflammatory molecules IL-8 and MMP-9 were down-regulated. These observed immune profiles are potentially reflective of the ability of the different groups to control SHIV replication. This study demonstrates that an optimized IL-15 immune adjuvant delivered with a DNA vaccine can impact the cellular immune profile in nonhuman primates and lead to enhanced suppression of viral replication.

Intramuscular and Intradermal Electroporation of HIV-1 PENNVAX-GP® DNA Vaccine and IL-12 Is Safe, Tolerable, Acceptable in Healthy Adults.

Background: Several techniques are under investigation to improve the immunogenicity of HIV-1 DNA vaccine candidates. DNA vaccines are advantageous due to their ease of design, expression of multiple antigens, and safety. METHODS: The HVTN 098 trial assessed the PENNVAX®-GP DNA vaccine (encoding HIV env, gag, pol) administered with or without plasmid IL-12 at 0-, 1-, 3-, and 6-month timepoints via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, adult participants. We report on safety, tolerability, and acceptability. RESULTS: HVTN 098 enrolled 94 participants: 85 received PENNVAX®-GP and nine received placebo. Visual analog scale (VAS) pain scores immediately after each vaccination were lower in the ID/EP than in the IM/EP group (medians 4.1-4.6 vs. 6-6.5, p < 0.01). IM/EP participants reported greater pain and/or tenderness at the injection site. Most ID/EP participants had skin lesions such as scabs/eschars, scars, and pigmentation changes, which resolved within 6 months in 51% of participants (24/55). Eighty-two percent of IM/EP and 92% of ID/EP participant survey responses showed acceptable levels of discomfort. CONCLUSIONS: ID/EP and IM/EP are distinct experiences; however, HIV-1 DNA vaccination by either route was safe, tolerable and acceptable by most study participants.

Robust antibody and cellular responses induced by DNA-only vaccination for HIV.

BACKGROUNDHVTN 098, a randomized, double-blind, placebo-controlled trial, evaluated the safety, tolerability, and immunogenicity of PENNVAX-GP HIV DNA vaccine, administered with or without plasmid IL-12 (pIL-12), via intradermal (ID) or intramuscular (IM) electroporation (EP) in healthy, HIV-uninfected adults. The study tested whether PENNVAX-GP delivered via ID/EP at one-fifth the dose could elicit equivalent immune responses to delivery via IM/EP and whether inclusion of pIL-12 provided additional benefit.METHODSParticipants received DNA encoding HIV-1 env/gag/pol in 3 groups: 1.6 mg ID (ID no IL-12 group, n = 20), 1.6 mg ID + 0.4 mg pIL-12 (ID + IL-12 group, n = 30), 8 mg IM + 1 mg pIL-12 (IM + IL-12 group, n = 30), or placebo (n = 9) via EP at 0, 1, 3, and 6 months. Results of cellular and humoral immunogenicity assessments are reported.RESULTSFollowing vaccination, the frequency of responders (response rate) to any HIV protein based on CD4+ T cells expressing IFN-γ or IL-2 was 96% for both the ID + IL-12 and IM + IL-12 groups; CD8+ T cell response rates were 64% and 44%, respectively. For ID delivery, the inclusion of pIL-12 increased CD4+ T cell response rate from 56% to 96%. The frequency of responders was similar (≥90%) for IgG binding antibody to gp140 consensus Env across all groups, but the magnitude was higher in the ID + IL-12 group compared with the IM + IL-12 group.CONCLUSIONPENNVAX-GP DNA induced robust cellular and humoral immune responses, demonstrating that immunogenicity of DNA vaccines can be enhanced by EP route and inclusion of pIL-12. ID/EP was dose sparing, inducing equivalent, or in some aspects superior, immune responses compared with IM/EP.TRIAL REGISTRATIONClinicalTrials.gov NCT02431767.FUNDINGThis work was supported by National Institute of Allergy and Infectious Diseases (NIAID), U.S. Public Health Service grants, an HIV Vaccine Design and Development Team contract, Integrated Preclinical/Clinical AIDS Vaccine Development Program, and an NIH award.

Sequential priming with simian immunodeficiency virus (SIV) DNA vaccines, with or without encoded cytokines, and a replicating adenovirus-SIV recombinant followed by protein boosting does not control a pathogenic SIVmac251 mucosal challenge.

Previously, combination DNA/nonreplicating adenovirus (Ad)- or poxvirus-vectored vaccines have strongly protected against SHIV(89.6P), DNAs expressing cytokines have modulated immunity elicited by DNA vaccines, and replication-competent Ad-recombinant priming and protein boosting has strongly protected against simian immunodeficiency virus (SIV) challenge. Here we evaluated a vaccine strategy composed of these promising components. Seven rhesus macaques per group were primed twice with multigenic SIV plasmid DNA with or without interleukin-12 (IL-12) DNA or IL-15 DNA. After a multigenic replicating Ad-SIV immunization, all groups received two booster immunizations with SIV gp140 and SIV Nef protein. Four control macaques received control DNA plasmids, empty Ad vector, and adjuvant. All vaccine components were immunogenic, but the cytokine DNAs had little effect. Macaques that received IL-15-DNA exhibited higher peak anti-Nef titers, a more rapid anti-Nef anamnestic response postchallenge, and expanded CD8(CM) T cells 2 weeks postchallenge compared to the DNA-only group. Other immune responses were indistinguishable between groups. Overall, no protection against intrarectal challenge with SIV(mac251) was observed, although immunized non-Mamu-A*01 macaques as a group exhibited a statistically significant 1-log decline in acute viremia compared to non-Mamu-A*01 controls. Possible factors contributing to the poor outcome include administration of cytokine DNAs to sites different from the Ad recombinants (intramuscular and intratracheal, respectively), too few DNA priming immunizations, a suboptimal DNA delivery method, failure to ensure delivery of SIV and cytokine plasmids to the same cell, and instability and short half-life of the IL-15 component. Future experiments should address these issues to determine if this combination approach is able to control a virulent SIV challenge.

Clinical and Immunologic Biomarkers for Histologic Regression of High-Grade Cervical Dysplasia and Clearance of HPV16 and HPV18 after Immunotherapy.

Purpose: As previously reported, treatment of high-grade cervical dysplasia with VGX-3100 resulted in complete histopathologic regression (CR) concomitant with elimination of HPV16/18 infection in 40.0% of VGX-3100-treated patients compared with only 14.3% in placebo recipients in a randomized phase IIb study. Here, we identify clinical and immunologic characteristics that either predicted or correlated with therapeutic benefit from VGX-3100 to identify parameters that might guide clinical decision-making for this disease.Experimental Design: We analyzed samples taken from cervical swabs, whole blood, and tissue biopsies/resections to determine correlates and predictors of treatment success.Results: At study entry, the presence of preexisting immunosuppressive factors such as FoxP3 and PD-L1 in cervical lesions showed no association with treatment outcome. The combination of HPV typing and cervical cytology following dosing was predictive for both histologic regression and elimination of detectable virus at the efficacy assessment 22 weeks later (negative predictive value 94%). Patients treated with VGX-3100 who had lesion regression had a statistically significant >2-fold increase in CD137+perforin+CD8+ T cells specific for the HPV genotype causing disease. Increases in cervical mucosal CD137+ and CD103+ infiltrates were observed only in treated patients. Perforin+ cell infiltrates were significantly increased >2-fold in cervical tissue only in treated patients who had histologic CR.Conclusions: Quantitative measures associated with an effector immune response to VGX-3100 antigens were associated with lesion regression. Consequently, these analyses indicate that certain immunologic responses associate with successful resolution of HPV-induced premalignancy, with particular emphasis on the upregulation of perforin in the immunotherapy-induced immune response. Clin Cancer Res; 24(2); 276-94. ©2017 AACR.

Soluble Factors Secreted by Endothelial Cells Allow for Productive and Latent HIV-1 Infection in Resting CD4+ T Cells.

In vitro, it is difficult to infect resting CD4+ T cells with human immunodeficiency virus type 1 (HIV), but infections readily occur in vivo. Endothelial cells (ECs) interact with resting CD4+ T cells in vivo, and we found previously that EC stimulation leads to productive and latent HIV infection of resting CD4+ T cells. In this study, we further characterize the interactions between EC and resting T cells. We found that resting CD4+ T cells did not require direct contact with EC for productive and/or latent infection to occur, indicating the involvement of soluble factors. Among 30 cytokines tested in a multiplex enzyme-linked immunosorbent assay (ELISA), we found that expressions for IL-6, IL-8, and CCL2 were much higher in EC-stimulated resting T cells than resting T cells cultured alone. IL-6 was found to be the soluble factor responsible for inducing productive infection of resting T cells, although direct contact with EC had an added effect. However, none of the cytokines tested, IL-6, IL-8, or CCL2, induced additional latent infection in resting T cells, suggesting that unidentified cytokines were involved. Intracellular molecules MURR1, c-Jun N-terminal kinase (JNK), and glucose transporter-1 (GLUT1) were previously shown in blocking HIV infection of resting CD4+ T cells. We found that the concentrations of these proteins were not significantly different in resting T cells before and after stimulation by EC; therefore, they are not likely involved in EC stimulation of resting CD4+ T cells, and a new mechanism is yet to be identified.

Immunogenicity testing of a novel engineered HIV-1 envelope gp140 DNA vaccine construct.

DNA vaccines expressing the envelope (env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating strong immune responses. In this study, we described the development of a recombinant plasmid DNA (pEK2P-B) expressing an engineered codon-optimized envelope gp140 gene of primary (nonrecombinant) HIV-1 subtype B isolate 6101. Codon usage and RNA optimization of HIV-1 structural genes has been shown to increase protein expression in vitro as well as in the context of DNA vaccines in vivo. To further increase the expression, a synthetic IgE leader with kozak sequences were fused into the env gene. The cytoplasmic tail of the gene was also truncated to prevent recycling. The expression of env by the recombinant pEK2P-B was evaluated using T7 coupled transcription/translation. The construct demonstrated high expression of the HIV-1 env gene in eukaryotic cells as demonstrated in transfected 293-T and RD cells. Immunogenicity of pEK2P-B was evaluated in mice using IFN-gamma ELISpot assay, and the construct was found to be highly immunogenic and crossreactive with HIV-1 clade C env peptides. Three immunodominant peptides were also mapped out. Furthermore, by performing a CFSE flow cytometry-based proliferation assay, 2.4 and 1.5% proliferation was observed in CD4+, CD8+, and CCR+ memory T cells, respectively. Therefore, this engineered synthetic optimized env DNA vaccine may be useful in DNA vaccine and other studies of HIV-1 immunogenicity.

Priming with plasmid DNAs expressing interleukin-12 and simian immunodeficiency virus gag enhances the immunogenicity and efficacy of an experimental AIDS vaccine based on recombinant vesicular stomatitis virus.

Of the various approaches being developed as prophylactic HIV vaccines, those based on a heterologous plasmid DNA prime, live vector boost vaccination regimen appear especially promising in the nonhuman primate/simian-human immunodeficiency virus (SHIV) challenge model. In this study, we sought to determine whether a series of intramuscular priming immunizations with a plasmid DNA vaccine expressing SIVgag p39, in combination with plasmid expressed rhesus IL-12, could effectively enhance the immunogenicity and postchallenge efficacy of two intranasal doses of recombinant vesicular stomatitis virus (rVSV)-based vectors expressing HIV-1 env 89.6P gp160 and SIVmac239 gag p55 in rhesus macaques. In macaques receiving the combination plasmid DNA prime, rVSV boost vaccination regimen we observed significantly increased SIVgag- specific cell-mediated and humoral immune responses and significantly lower viral loads postintravenous SHIV89.6P challenge relative to macaques receiving only the rVSV vectored immunizations. In addition, the plasmid DNA prime, rVSV boost vaccination regimen also tended to increase the preservation of peripheral blood CD4+ cells and reduce the morbidity and mortality associated with SHIV89.6P infection. An analysis of immune correlates of protection after SHIV89.6P challenge revealed that the prechallenge SHIV-specific IFN-gamma ELISpot response elicited by vaccination and the ability of the host to mount a virus-specific neutralizing antibody response postchallenge correlated with postchallenge clinical outcome. The correlation between vaccine-elicited cell-mediated immune responses and an improved clinical outcome after SHIV challenge provides strong justification for the continued development of a cytokine-enhanced plasmid DNA prime, rVSV vector boost immunization regimen for the prevention of HIV infection.

T-cell responses induced in normal volunteers immunized with a DNA-based vaccine containing HIV-1 env and rev.

OBJECTIVE: An effective HIV-1 vaccine will likely need to induce strong cell-mediated immunity in humans. Therefore, we examined the ability of a DNA HIV-1 vaccine to induce a T-cell response in HIV-1 seronegative humans. DESIGN: Individuals were enrolled in a phase I clinical trial of safety and immune responses to an env/rev-containing plasmid at doses of 100, 300 or 1000 microg. Peripheral blood mononuclear cells (PBMC) samples were analyzed by standard lymphocyte proliferation, cytotoxic T lymphocyte (CTL) and ELISPOT techniques. RESULTS: PBMCs from subjects immunized with doses as low as 300 microg proliferated in vitro to env (four of six) or (three of six) proteins. Importantly, when the dose of vaccine was increased to 1000 microg of DNA, lymphocytes secreted IFN-gamma in an ELISPOT assay following in vitro stimulation with env (three of six) or rev (four of six) proteins. CONCLUSION: We observed HIV-1 DNA plasmid vaccines induce CD4 T-helper cell responses in humans. We observed a discrepancy in the CD4 versus CD8 response suggesting the importance of analyzing both compartments in clinical evaluation. Furthermore, this report demonstrates the high level of immunogenicity of and its importance as a component of a prophylactic vaccine for HIV-1.

Vaccine development against HIV-1: current perspectives and future directions.

The development of an efficacious vaccine against the human immunodeficiency virus (HIV) is of great urgency, because it is accepted that vaccination is the only means capable of controlling the AIDS pandemic. The foundation of HIV vaccine development is the analysis of immune responses during natural infection and the utilization of this knowledge for the development of protective immunization strategies. Initial vaccine development and experimentation are usually in animal models, including murine, feline, and nonhuman primates. Experimental vaccine candidates are closely studied for both efficacy and safety before proceeding to human clinical trials. There are a number of different therapeutic and prophylactic vaccine strategies currently being studied in human clinical trials. Vaccine strategies that are being tested, or have previously been tested, in humans include subunit, DNA plasmid, and viral vector, and combinations of these various strategies. Some of the results of these trials are promising, and additional research has focused on the development of appropriate chemical and genetic adjuvants as well as methods of vaccine delivery to improve the host immune response. This review summarizes the vaccine strategies that have been tested in both animal models and human clinical trials.