Earlier work in a number of disease models has shown that immunogens containing tandemly repeated epitopes can be more immunogenic than a respective immunogen containing only a single copy of the sequence. The applicant's studies have revealed that antisera raised in mice to a recombinant immunogen in alum containing eight copies of an MN isolate V3PND sequence is able to neutralize ADA, a primary M-tropic isolate of HIV-I. However, despite hopes of generating immunogens able to induce antibodies capable of primary isolate neutralization, the hypervariable nature of the V3 loop of HIV would apparently necessitate incorporation of multiple strain specific V3 loop sequences in any prototype AIDS vaccine. Coincident with the increased understanding of the role of the V3 loop in determining cellular tropism, however, has come new insights into how the V3loop sequences interact with cellular coreceptors in the facilitation of HIV infection. CCR5 is a G-protein coupled receptor identified as integral for entry by M-tropic HIV isolates into macrophages. Since M-tropic viral isolates are especially implicated early in HIV infection and individuals who are homozygous for the 32 bp deletion which results in a non-functional-CCR5 receptor are protected from infection from HIV, yet appear to have a normal phenotype, the CCR5 extracellular receptor sequences represent attractive targets for humoral immunity to HIV-I. We have analyzed the CCR5 extracellular receptor sequence domains and have identified putative extracellular sequences which represent potential antibody targets. The major objectives of the current study are to: a) Develop tandem repeat soluble and DNA constructs representing sequences from the V3 loop of primary NSI M-tropic isolates; b) Analyze the ability of antisera to these constructs raised in BALB/c mice to neutralize homologous and heterologous primary NSI M-tropic HIV isolates; c) Develop tandem repeat soluble and DNA constructs representing sequences from the putative extracellular domains of CCR5; d) Analyze the ability of antisera to these constructs raised in BALB/c mice to neutralize homologous and heterologous primary NSI M-tropic HIV isolates and; e)Utilizing the results from targeting the V3 and CCR5 sequences, construct hybrid immunogens which contain sequences from both the V3 loop of primary NSI M-tropic viruses, and immunogenic sequences from the CCR5 coreceptor. The construction of these immunogens is enabled by our development of BMX7, a novel vector for cloning multi-determinant tandem repeat immunogens, and BMX7EX, a DNA expression vector which retains the essential cloning features of the BMX7 vector. These studies will ideally culminate in the development of immunogens capable of inducing antibodies able to neutralize M-tropic primary isolate infectivity and contribute significantly to our understanding of the immunology of peptide component DNA and soluble immunogens.
