Effective DNA-immunogens based on the consensus protease of the HIV-1 clade A strain predominant in the territory of the former Soviet Union

Maria Isaguliants¹, Athina Kilpeläinen¹, Stefan Petkov¹, Anastasia Latanova¹,², Oleg Latishev², Elizaveta Starodubova¹,3
Affiliates: ¹Department of Microbiology,Tumor and Cell Biology, Karolinska Institutet, Stockholm, Sweden, ²D.I. Ivanovsky Institute of Virology, Moscow, Russia, 3W.A. Engelhard Institute of Molecular Biology, Moscow, Russia3

Background
Highly active antiretroviral therapy limits HIV-1 load and spread, but drug shortages and poor therapy compliance promote emergence and spread of drug-resistant (DR) strains limiting therapeutic options. An immunization against DR HIV in combination with antiviral therapy could limit the development of drug-resistance and prolong the effective treatment. Here, we designed and pre-clinically tested the components for such treatment targeting HIV protease (PR).

Methods
PR sequences from treatment-naïve patients infected with the FSU-A HIV-1 strain, predominant in the territory of the former Soviet Union (FSU) were selected, aligned and used to create a consensus PR (PR_A). A humanized PR_A gene was synthesized and cloned for pro- and eukaryotic expression. Mutations of resistance to protease inhibitors in positions 46, 54, and 82 and inactivation mutation in position 25 were introduced by site-directed mutagenesis. Expression of PR_A variants in E. coli and in HeLa cells was confirmed by Western blot using in-house polyclonal rabbit antibodies against PR_A. Expression levels were assessed in the oocytes of Xenopus laevis by evaluating 14C-labeled Lysine incorporation into newly synthesized proteins. Genes for active and inactive PR in pVax1 were used to immunize BALB/c mice by intradermal injection followed by electroporation. Cellular responses were assessed as the capacity of murine splenocytes to recognize peptides derived from PR_A, PR_B and PR_A with drug-resistance mutations as registered by IFN-γ/IL-2 Fluorospot.

Results
Two consensus sequences were generated from the sequences originating from the FSU countries, 1st s set dated 1996-2003 (n=218) and 2nd, 2000-2011 (n=40). Consensus sequences were identical reflecting low genetic diversity of the FSU-A strain. Humanized gene of PR_A was synthesized and mutated to encode inactive PR_A (D25N; PR_Ai) and active and inactive DR PR variants harboring M46I, I54V, V82A. All synthetic genes encoded protein with expected molecular mass of 11 kDa specifically stained by rabbit antibodies against HIV PR. Proteins were proteolytically unstable, and accumulated only after treatment of expressing cells with proteasomal inhibitor MG132. PR_A and its DR variants were enzymatically active. Introduction of D25N led to 80% loss of their activity in HIV-1 protease activity assays both in the lysates of PR-expressing HeLa and in the oocytes. At the same time, insertion of D25N led to a 5-fold increase in PR expression as compared to the respective active variants. PR_A and  PR_Ai genes were used to immunize BALB/C mice. They were compared to the genes for active and inactive PR of HXB2 (PR_B, PR_Bi; Hallengärd D et al, 2011). Cellular immunogenicity of PR genes increased in a row PR_B < PR_Bi ≤  PR_A < PR_Ai. DNA-immunization with the PR_Ai induced the strongest cytokine secretion in response to all PR peptides tested. Genes induced cellular responses against the wild-type peptides, but not their analogues carrying DR mutations indicating that DR mutations alter PR epitopes.

Conclusions
Combination of expression optimization and proteasomal degradation provides for potent performance of the consensus HIV-1 clade A protease in gene immunization. As DR mutations alter PR epitopes, their acquisition by the virus may lead to an immune escape. Immune response against DR HIV-1 would therefore require drug resistant forms of the viral antigens. PR_A variants obtained here could serve as a platform for such immunization.