A Novel Liposome-Based Adjuvant CAF01 for Induction of CD8+ Cytotoxic T-Lymphocytes (CTL) to HIV-1 Minimal CTL Peptides in HLA-A*0201 Transgenic Mice

Background: Specific cellular cytotoxic immune responses (CTL) are important in combating viral diseases and a highly desirable feature in the development of targeted HIV vaccines. Adjuvants are key components in vaccines and may assist the HIV immunogens in inducing the desired CTL responses. In search for appropriate adjuvants for CD8+ T cells it is important to measure the necessary immunological features e.g. functional cell killing/lysis in addition to immunological markers that can be monitored by simple immunological laboratory methods. Methodology/Principal Findings: We tested the ability of a novel two component adjuvant, CAF01, consisting of the immune stimulating synthetic glycolipid TDB (Trehalose-Dibehenate) incorporated into cationic DDA (Dimethyldioctade-cylammonium bromide) liposomes to induce CD8+ T-cell restricted cellular immune responses towards subdominant minimal HLA-A0201-restricted CTL epitopes from HIV-1 proteins in HLA-A*0201 transgenic HHD mice. CAF01 has an acceptable safety profile and is used in preclinical development of vaccines against HIV-1, malaria and tuberculosis. Conclusions/Significance: We found that CAF01 induced cellular immune responses against HIV-1 minimal CTL epitopes in HLA-A*0201 transgenic mice to levels comparable with that of incomplete Freund’s adjuvant

Immunological analysis of a Lactococcus lactis-based DNA vaccine expressing HIV gp120

For reasons of efficiency Escherichia coli is used today as the microbial factory for production of plasmid DNA vaccines. To avoid hazardous antibiotic resistance genes and endotoxins from plasmid systems used nowadays, we have developed a system based on the food-grade Lactococcus lactis and a plasmid without antibiotic resistance genes. We compared the L. lactis system to a traditional one in E. coli using identical vaccine constructs encoding the gp120 of HIV-1. Transfection studies showed comparable gp120 expression levels using both vector systems. Intramuscular immunization of mice with L. lactis vectors developed comparable gp120 antibody titers as mice receiving E. coli vectors. In contrast, the induction of the cytolytic response was lower using the L. lactis vector. Inclusion of CpG motifs in the plasmids increased T-cell activation more when the E. coli rather than the L. lactis vector was used. This could be due to the different DNA content of the vector backbones. Interestingly, stimulation of splenocytes showed higher adjuvant effect of the L. lactis plasmid. The study suggests the developed L. lactis plasmid system as new alternative DNA vaccine system with improved safety features. The different immune inducing properties using similar gene expression units, but different vector backbones and production hosts give information of the adjuvant role of the silent plasmid backbone. The results also show that correlation between the in vitro adjuvanticity of plasmid DNA and its capacity to induce cellular and humoral immune responses in mice is not straight forward

Cellular immune responses induced after immunization in CAF01 with or without a T helper epitope.

<p>IFN-γ ELISPOT responses against (A) Vif₁₀₁ CTL epitope and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE (grey bars, n = 5) or with Vif₁₀₁ alone (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from individual mice immunized with (C) Vif₁₀₁ and PADRE (grey, n = 5) or (D) with Vif₁₀₁ alone (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant levels are considered >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Cellular immune responses are induced to a similar level after immunization with IFA and CAF01.

<p>IFN-γ ELISPOT responses against (A) HLA-A2-restricted CD8 T cell epitope Vif₁₀₁ and (B) PADRE Th epitope of splenocytes from HLA-A*0201 transgenic HHD mice after 10 days of s.c. immunization with Vif₁₀₁ and PADRE in either IFA (grey bars, n = 5) or CAF01 adjuvants (black bars, n = 5) and 5 days <i>in vitro</i> prestimulation with individual epitopes. The background of an unimmunized mouse is substracted. Specific ⁵¹Cr-release from target cells preloaded with Vif₁₀₁ after incubation with effector splenocytes from Vif₁₀₁ and PADRE immunized mice adjuvanted with (C) IFA (grey, n = 5) or (D) CAF01 (black, n = 5). The percentage of specific lysis was calculated as 100×(experimental release-spontaneous release)/(total release-spontaneous release). Background lysis from an unimmunized mouse is shown (open circles). Significant positive levels are considered for >10% lysis at a 50∶1 ratio of effector:target (E:T) cells. One representative experiment out of three. SFU, spot forming units.</p

Proliferative CD8⁺ T cell responses induced after immunization with IFA and CAF01.

<p>The percentage of proliferating CD3+CD8⁺ splenocytes (having reduced CFSE dye) derived from HLA-A*0201 transgenic HHD mice 10 days after s.c. immunization with a peptide mix consisting of 10 peptides adjuvanted with (A) IFA or (B) CAF01. The background proliferation of non-stimulated (NS) cells, incubated with media alone is shown. The epitope peptides were (from left to right): NS, Gag₁₅₀, Gag₄₃₃, Env₆₇, Pol₆₀₆, Vpu₆₆, Vif₁₀₁, Vif₂₃, Gag₂₉₈ (Th), Env₅₇₀ (Th) and PADRE (Th).</p