The best known correlate of protection provided by vaccines is the presence of pathogen specific antibodies after immunization. However, against the Human Immunodeficiency Virus-1 (HIV-1) the mere presence of antibodies specific for the viral Envelope (Env) protein is not sufficient to provide protection. This necessitates in depth study of the humoral responses elicited during infection and by vaccination. While a significant amount of effort has been invested in studying the evolution of antibody responses to viral infection, only limited progress in understanding antibody responses elicited through vaccination has been made. In the studies described here, I attempt to rectify this deficiency by investigating how the quality of a humoral response is altered with the use of different immunization regimens, in particular a DNA prime-protein boost regimen, or with the use of different model HIV-1 Env gp120 immunogens. In a New Zealand White (NZW) rabbit model, we demonstrate that the broader neutralizing activity elicited with the DNA prime-protein boost regimen may be the result of the elicitation of a higher avidity antibody response and a unique profile of antibody specificities. Specifically, use of a DNA prime-protein boost regimen elicits antibodies targeted to the CD4 binding domain of the HIV-1 Env, a specificity that was not frequently observed when only protein based immunizations were administered. We extended this analysis to sera from healthy human volunteers who participated in early phase HIV vaccine trials utilizing either a protein alone immunization regimen, a canarypox prime-protein boost immunization regimen, or a DNA prime-protein boost immunization regimen. Evaluation of sera from these trials demonstrated that the use of a DNA prime-protein boost regimen results in an antibody response with greater neutralization breadth characterized by an increased frequency and titer of antibodies targeted toward the CD4 binding site (CD4bs). In addition to this, the antibody response elicited by the DNA prime-protein boost regimen also exhibited the capability to mediate antibody dependent cell-mediated cytotoxicity (ADCC) activity as well as activation of the complement system. Additionally, in an attempt to better understand the capabilities of antibodies elicited by a DNA prime-protein boost regimen, we generated gp120 specific monoclonal antibodies (mAbs) from a single DNA primed-protein boosted NZW rabbit. Analysis of mAbs produced from this animal revealed that use of this immunization regimen elicits an antibody repertoire with diverse epitope specificity and cross reactivity. Furthermore, these select mAbs are capable of neutralizing heterologous HIV isolates. Further application of mAb generation in rabbits may provide a valuable tool to study immunogenicity of different vaccines and immunization regimens. Concurrently, while demonstrating that a DNA prime-protein boost regimen elicits a higher quality antibody response than that observed with other leading techniques, we also demonstrated that immunogen selection can play a vital role in the quality of the resulting antibody response. By immunizing with two closely related but phenotypically distinct model gp120 immunogens, known as B33 and LN40, we demonstrated that disparate gp120s have different intrinsic abilities to raise a heterologous neutralizing antibody response. Additionally, we showed that residues found within and flanking the b12 and CD4 binding sites play critical roles in modulating neutralizing activity of sera from animals immunized with LN40 gp120, indicating that the broader neutralizing activity seen with this immunogen may be due to differential elicitation of antibodies to this domain.

Around thirty years ago HIV-1 was identified, and from that point the known epidemic has grown to over 30 million infected individuals. Early on in the course of HIV-1 research, viruses were classified as either syncytia inducing, CXCR4-using, T-cell tropic or non-syncytia inducing, CCR5-using, macrophage tropic. Since that time, several groups have shown that this is an oversimplification. There is a great deal of diversity amongst CCR5-using HIV-1 variants. There remains a great deal to be discovered regarding HIV-1 CCR5-tropism and how this affects other aspects of HIV-1 infection. The CD4 binding site (CD4bs) on the HIV-1 envelope plays a major role in determining the capacity of R5 viruses to infect primary macrophages. Thus, envelope determinants within or proximal to the CD4bs have been shown to control the use of low CD4 levels on macrophages for infection. These residues affect the affinity for CD4 either directly or indirectly by altering the exposure of CD4 contact residues. In this thesis, a single amino acid determinant is described in the V1 loop that also modulates macrophage tropism. I identified an E153G substitution that conferred high levels of macrophage infectivity for several heterologous R5 envelopes, while the reciprocal G153E substitution abrogated infection. Shifts in macrophage tropism were associated with dramatic shifts in sensitivity to the V3 loop monoclonal antibody (MAb), 447-52D and soluble CD4, as well as more modest changes in sensitivity to the CD4bs MAb, b12. These observations are consistent with an altered conformation or exposure of the V3 loop that enables the envelope to use low CD4 levels for infection. The modest shifts in b12 sensitivity suggest that residue 153 impacts on the exposure of the CD4bs. However, the more intense shifts in sCD4 sensitivity suggest additional mechanisms that likely include an increased ability of the envelope to undergo conformational changes following binding to suboptimal levels of cell surface CD4. In summary, a conserved determinant in the V1 loop modulates the V3 loop to prime low CD4 use and macrophage infection. In addition to determinants, this thesis seeks to evaluate the roles of macrophage tropic and non-macrophage tropic envelopes during the course of infection. Non-macrophage tropic virus predominates in immune tissue throughout infection, even in individuals suffering from HIV-associated dementia (HAD) who are known to carry many macrophage tropic viruses. There must be some advantage for these non-macrophage tropic viruses allowing them to persist in immune tissue throughout the disease. This thesis demonstrates that there is no advantage for these viruses to directly infect CD4+ T-cells, nor is there an advantage for them to be preferentially transmitted by dendritic cells to CD4+ T-cells. Given that transmitted/founder (T/F) viruses may preferentially interact with α4β7, and T/F viruses are non-macrophage tropic, I tested whether non-mac viruses could utilize α4β7 to their advantage. These experiments show that macrophage tropism does not play a role in gp120 interactions with α4β7. I evaluated whether there was a distinct disadvantage to macrophage tropic Envs, given their ability to infect dendritic cells and possibly stimulate the innate immune response. Using infected monocyte-derived dendritic cells (MDDCs), it was shown that mac-tropic Envs do not generate a significant immune response. These experiments demonstrate that there does not appear to be any advantage to non-macrophage tropic Envs, and that macrophage tropic Envs are able to infect CD4+ T-cells more efficiently, as well as DCs.