We have established that heat-killed Brucella abortus conjugated to a V3-loop peptide from HIV-1 MN can elicit neutralizing antibodies and CTL in normal and mice depleted of CD4+ T-cells, systemically and at mucosal surfaces. Furthermore this conjugate can induce neutralizing antibody and cellular responses in Rhesus macaques. The peptide used in these studies bears single CTL and B cell epitopes. As such it was possible to use this peptide in inbred mice to obtain proof of concept for the attributes of B. abortus as a carrier capable of inducing CD4+ T-cell independent B cell and CTL responses. However, such a system could not be expected to (i) elicit antibodies to multiple isolates belonging to different clades, and (ii) generate CTL in an outbred population. As a first step in accomplishing these aims it is necessary to determine whether B. abortus can also act as a carrier for more complex proteins such as envelope and structural proteins from HIV-1. Inactivated but not denatured HIV-1 viral particles will also be available for these studies and have the advantage of presenting viral proteins in the native form. In the case of viral proteins and particles, which unlike small peptides may be immunogenic by themselves, it may be possible to elicit responses using mixtures with B. abortus, rather than using conjugates. That is using B. abortus as an adjuvant rather than as a carrier. Preliminary results show that conjugates of gp-120 to B. abortus can induce neutralizing antibodies in mice and that these are more cross-reactive than antibodies elicited by B. abortus-peptide conjugates. These experiments will be repeated to study systemic and mucosal response in normal mice and in mice lacking CD4+ T-cells. Antigen-specific cellular responses as well as antibody responses will be assessed. These will include generation of IFNgamma-secreting CD8+ cells and CTL. Similar experiments will be performed in Rhesus macaques. We have established that heat-killed Brucella abortus conjugated to a V3-loop peptide from HIV-1 MN can elicit neutralizing antibodies and CTL in normal and mice depleted of CD4+ T-cells, systemically and at mucosal surfaces. The antibodies elicited in mice lacking CD4+ T-cells were mainly of the IgG2a isotype. Sera from these mice were more efficient than sera containing predominantly IgG1 in neutralizing HIV-1. Furthermore this conjugate can induce neutralizing antibody (systemic and mucosal) and cellular responses in Rhesus macaques. The peptide used in these studies bears single CTL and B cell epitopes. In future studies chemically inactivated viruses that retain their native structure will be used to obtain a more diverse immune response. Previously, we have shown that a strong Th1 stimulus, in the form of Brucella abortus, can switch Th2 to Th1 responses and inhibit IgE induction, i.e suppress an IL-4-dependent allergic response. These studies were performed in adult mice. In order to determine whether this approach may be useful in immature mice, we studied neonates in terms of antibody isotypes and cytokines following exposure to an antigen, ovalbumin, in the absence or in the presence of B. abortus. We found that neonatal mice can be primed to ovalbumin (ova) and will make IgE responses as young adults if boosted with ova. This IgE response was inhibited if the neonates were exposed to B. abortus at the time of ova inoculation. This result has implications for treatment of neonates with foreign proteins (e,g, treatment of hemophiliacs with factor VIII). Avoidance of unwanted antibody responses that are dependent on IL-4 can be achieved by concomitant administration of a strong Th1 stimulus. Mouse IgG2a is a complement-fixing isotype. In order to study whether human complement-fixing antibodies are more potent in neutralizing virus, polyclonal antibody has been obtained from individuals with high titer against respiratory syncytia virus (RSVIG). This product was passed over protein A columns and purified into IgG3 and enriched for IgG2, IgG1 and IgG4. Further purifications will involve immunoaffinity columns. The purified antibodies will be titrated for potency in a RSV neutralization assay. These results will determine whether different human isotypes are more active than others in antiviral activity. By performing mixing experiments it should b possible to study whether non complement-fixing antibodies exhibit blocking or inhibitory activity.
