HIV-1 DNA VACCINES THAT TARGET AND ACTIVATE CD40

HIV-1 DNA疫苗在实验动物中诱导体液和细胞免疫反应，这表明它们在免疫方面具有潜在的用途。然而，需要进一步优化DNA疫苗，以诱导对强毒HIV-1毒株的保护。我们建议利用CD40的激活和靶向来尝试提高HIV-1包膜DNA疫苗产生强大的细胞免疫和高滴度中和抗体的能力。我们将构建编码CD40配体(CD154)或针对CD40的单链抗体(ScFv)的DNA疫苗，与HIV-1包膜蛋白编码部分的DNA融合。DNA免疫后，该融合蛋白在体内的表达可导致CD40受体的激活和HIV-1包膜抗原进入CD40阳性抗原提呈细胞(APC)的胞内途径。与CD40阳性抗原提呈细胞(APC)结合后env抗原内化。在与CD40受体结合后，env抗原的内化将增强多肽的呈递，产生有效的抗体，并产生CD4+辅助T细胞和CD8+CTL活性。我们推测，CD40激活和MHC第二类呈递病毒多肽的结合将导致对病毒抗原的免疫反应增强。这一假设将通过构建抗人CD40单链抗体-env DNA疫苗来验证，然后在灵长类动物中进行测试，其中抗人CD40单链抗体和人CD154保持CD40结合和功能活性。针对HIV-1 env的抗体反应将通过表位特异性的ELISA法和病毒中和法进行检测。

HIV-1 DNA vaccines induce both humoral and cellular immune responses in experimental animals, suggesting their potential usefulness for immunization. However, further optimization of DNA vaccines is needed to induce protection against virulent HIV-1 isolates. We propose to utilize CD40 activation and targeting in an attempt to improve the ability of HIV-1 env DNA vaccines to generate strong cellular immunity and high titers of neutralizing antibodies. We will construct DNA vaccines encoding CD40 ligand (CD154) or a single chain Fv (scFv) specific for CD40, fused with DNA encoding portions of the HIV-1 env protein. After DNA vaccination, the expression of this fusion protein in vivo could result in both activation of the CD40 receptor and direction of HIV-1 env antigens into the endocytic pathway of CD40 positive antigen presenting cells (APC). Internalization of env antigens after binding the CD40 positive antigens presenting cells (APC). Internalization of env antigens after binding the CD40 receptor will enhance presentation of peptides to effective antibody production and generation of CD4+ helper T cell and CD8+CTL activity. We hypothesize that the combination of CD40 activation and presentation of viral peptides by MHC class II will lead to improved immune responses to viral antigens. This hypothesis will be tested by construction of anti-human CD40 scFv-env DNA vaccines, followed by testing in primates where the anti-human CD40 scFv and human CD154 retain CD40 binding and functional activity. Antibody responses towards HIV-1 env will be tested by epitope specific ELISA assays and viral neutralization assays.