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CD8+ cytotoxic T lymphocytes (CTLs) are critical for clearing many viral infections, and protective CTL memory can be induced by vaccination with attenuated viruses and vectors. Non-replicating vaccines are typically potentiated by the addition of adjuvants that enhance humoral responses, however few are capable of generating CTL responses. Adjuplex is a carbomer-lecithin-based adjuvant demonstrated to elicit robust humoral immunity to non-replicating antigens. We report that mice immunized with non-replicating Adjuplex-adjuvanted vaccines generated robust antigen-specific CTL responses. Vaccination by the subcutaneous or the intranasal route stimulated systemic and mucosal CTL memory respectively. However, only CTL memory induced by intranasal vaccination was protective against influenza viral challenge, and correlated with an enhancement of memory CTLs in the airways and CD103+ CD69+ CXCR3+ resident memory-like CTLs in the lungs. Mechanistically, Myd88-deficient mice mounted primary CTL responses to Adjuplex vaccines that were similar in magnitude to wild-type mice, but exhibited altered differentiation of effector cell subsets. Immune potentiating effects of Adjuplex entailed alterations in the frequency of antigen-presenting-cell subsets in vaccine draining lymph nodes, and in the lungs and airways following intranasal vaccination. Further, Adjuplex enhanced the ability of dendritic cells to promote antigen-induced proliferation of naïve CD8 T cells by modulating antigen uptake, its intracellular localization, and rate of processing. Taken together, we have identified an adjuvant that elicits both systemic and mucosal CTL memory to non-replicating antigens, and engenders protective CTL-based heterosubtypic immunity to influenza A virus in the respiratory tract. Further, findings presented in this manuscript have provided key insights into the mechanisms and factors that govern the induction and programming of systemic and protective memory CTLs in the respiratory tract., Author Summary Current respiratory-virus vaccines typically employ non-replicating antigens and rely solely on the generation of humoral responses for protection. Viruses such as influenza can mutate and escape these responses, thereby limiting immunity and necessitating revaccination. Cell-mediated immunity (CMI) could provide broader protection by targeting viral components that infrequently mutate, however non-replicating vaccines capable of inducing CMI are not available. Impediments to vaccine development include an incomplete understanding of the nature of protective respiratory CMI and a lack of vaccine adjuvants capable of eliciting CMI to non-replicating antigens. Using a mouse model, we characterized the protective immunity afforded by CMI responses to non-replicating vaccines formulated with the adjuvant Adjuplex. We found that vaccination via either the subcutaneous or intranasal route was capable of inducing potent CMI responses. However, only intranasal vaccination protected against challenge with heterosubtypic influenza viruses. This protection correlated with enhancement of T cells with a resident-memory phenotype in the lungs. Additionally, mechanistic studies showed that Adjuplex affects antigen-presenting cells via activation and alteration of antigen uptake, processing, and presentation. The current studies: (1) identified an adjuvant that elicits protective CMI to respiratory viral pathogens; (2) suggested that stimulation of protective CMI in the respiratory tract requires intranasal vaccine delivery.

The development of a rapid and efficient system to identify human immunodeficiency virus type 1 (HIV-1)-infected individuals with broad and potent HIV-1-specific neutralizing antibody responses is an important step toward the discovery of critical neutralization targets for rational AIDS vaccine design. In this study, samples from HIV-1-infected volunteers from diverse epidemiological regions were screened for neutralization responses using pseudovirus panels composed of clades A, B, C, and D and circulating recombinant forms (CRFs). Initially, 463 serum and plasma samples from Australia, Rwanda, Uganda, the United Kingdom, and Zambia were screened to explore neutralization patterns and selection ranking algorithms. Samples were identified that neutralized representative isolates from at least four clade/CRF groups with titers above prespecified thresholds and ranked based on a weighted average of their log-transformed neutralization titers. Linear regression methods selected a five-pseudovirus subset, representing clades A, B, and C and one CRF01_AE, that could identify top-ranking samples with 50% inhibitory concentration (IC50) neutralization titers of ≥100 to multiple isolates within at least four clade groups. This reduced panel was then used to screen 1,234 new samples from the Ivory Coast, Kenya, South Africa, Thailand, and the United States, and 1% were identified as elite neutralizers. Elite activity is defined as the ability to neutralize, on average, more than one pseudovirus at an IC50titer of 300 within a clade group and across at least four clade groups. These elite neutralizers provide promising starting material for the isolation of broadly neutralizing monoclonal antibodies to assist in HIV-1 vaccine design.

Despite repeated exposure to HIV-1, certain individuals remain persistently uninfected. Such exposed uninfected (EU) people show evidence of HIV-1–specific T cell immunity and, in rare cases, selective resistance to infection by macrophage-tropic strains of HIV-1. The latter has been associated with a 32–base pair deletion in the C–C chemokine receptor gene CCR-5, the major coreceptor of macrophage-tropic strains of HIV-1. We have undertaken an analysis of the HIV-specific T cell responses in 12 EU individuals who were either homozygous for the wild-type CCR-5 allele or heterozygous for the deletion allele (CCR-5Δ32). We have found evidence of an oligoclonal T cell response mediated by helper T cells specific for a conserved region of the HIV-1 envelope. These cells produce very high levels of C–C chemokines when stimulated by the specific antigen and suppress selectively the replication of macrophage-tropic, but not T cell–tropic, strains of HIV-1. These chemokine-producing helper cells may be part of a protective immune response that could be potentially exploited for vaccine development.

Neutralization of HIV-1 in vitro by anti-HLA class I antibodies suggests that class I molecules are involved in HIV-1 infection. HIV-infected cells can fuse with uninfected cells in a process that leads to the formation of multinucleated syncytia, involving an interaction between host and viral antigens expressed at the cell surfaces. We used a syncytium assay between the 8E5 cell line chronically infected with a pol-defective variant of LAV IIIb, and the CD4-positive cell line MOLT3, to study the role of HLA class I in HIV-1-induced cell fusion. By probing cells with a panel of anti-HLA monoclonal antibodies (MABs) we demonstrated that the fusion process is modulated specifically by C alleles of HLA class I expressed on uninfected cells but not by that on already infected cells. Addition of beta 2-microglobulin to the cocultures resulted in a dose-dependent enhancement in both the number and size of syncytia, whereas exogenous HLA-C-restricted peptides inhibited syncytium formation, implying that only certain conformational states of HLA class I are permissive for syncytium formation. Treatment of cocultures with HLA-Cw4-restricted peptides containing amino acid substitutions in the anchor residues showed that syncytium inhibition was dependent on conventional binding of the peptide inside the groove. The data indicate that HLA class I, in a conformation free of peptide but associated with beta 2-microglobulin, can directly influence virus-induced cell fusion.

A growing number of reports indicates that certain groups of individuals who almost certainly have been exposed to human immunodeficiency virus (HIV), yet continue to exhibit no signs or symptoms of infection, often have subtle evidence of specific immunity. We studied such a high-risk (HR) cohort of persistently seronegative individuals with histories of long-term sexual exposure to an HIV-infected partner to look for evidence of both humoral and cellular immunity that might have been induced by exposure to the virus. Twenty-three heterosexual and four homosexual monogamous couples with discordant HIV status were included in the study. Twelve of the HR partners were studied for in vitro stimulation of peripheral blood mononuclear cells (PBMC) by HIV envelope-derived peptides. All 12 responded overwhelmingly to a peptide containing the fifth conserved region of gp120. By generating and cloning T cell lines specific for this peptide, we concluded that in these individuals the T cell response to the envelope is mainly focused on the carboxy-terminus region of gp120 and is characterized by an oligoclonal expansion of CD4+ T cells expressing the same TCR. Eighteen HR partners and 37 HIV-1 seropositive subjects were tested for the presence of anti-CD4 antibodies (anti-CD4 Abs) using a recombinant CD4-based enzyme-linked immunosorbent assay (ELISA). Anti-CD4 Abs were detected in eight of the HR partners (six confirmed by Western blot) and in nine of the HIV-1 seropositive subjects (eight confirmed by Western blot). Results from binding competition assays with a panel of monoclonal anti-CD4 Abs suggested that the anti-CD4 Abs detected in the HR partners are directed toward epitopes that are induced by gp120 binding. Twenty-seven of the HR partners were tested for the presence of antibodies that cross-react with HLA class I and gp120 (anti-HLA Abs). Anti-HLA Abs were detected in 16 of the HR partner sera and in 4 94 sera from a control population of normal healthy blood donors. Taken together, the results suggest that in some individuals with a history of long-term exposure to HIV, specific immunity may develop in the absence of overt infection. The common trigger for these responses is gp120.

Female C3H/HeJ mice were immunized parenterally and/or intragastrically with two important food allergens, soy and shrimp. Although both preparations elicited specific IgE production when administered intraperitoneally, anti-shrimp titers were consistently higher. Soy or shrimp administration, i.g. with Pertussis adjuvant (intraperitoneally or intragastrically) did not induce detectable reaginic antibody. Animals treated with soy intragastrically were unable to produce a soy-specific IgE response upon intraperitoneal immunization. The combination of soy and Pertussis (intragastric) did, however, abrogate this tolerizing effect on IgE synthesis. In contrast to soy, the shrimp antigens (intragastric) did not act as tolerogens and were found to enhance subsequent reaginic responses produced by intraperitoneal immunization. These results are considered with respect to mechanisms operative in food allergy.