Transfer of HIV-1-specific cytotoxic T lymphocytes to an AIDS patient leads to selection for mutant HIV variants and subsequent disease progression.

An HIV-1-seropositive volunteer was infused with an expanded autologous cytotoxic T lymphocyte (CTL) clone directed against the HIV-1 nef protein. This clone was adoptively transferred to determine whether supplementing CTL activity could reduce viral load or improve clinical course. Unexpectedly, infusion was followed by a decline in circulating CD4+ T cells and a rise in viral load. Some of the HIV isolates obtained from the plasma or CD4+ cells of the patient were lacking the nef epitope. These results suggest that active CTL selection of viral variants could contribute to the pathogenesis of AIDS and that clinical progression can occur despite high levels of circulating HIV-1-specific CTLs.

Characterization of MHC class I restricted cytotoxic T cell responses to tax in HTLV-1 infected patients with neurologic disease.

To understand the nature of the cytotoxic T cell response generated in human T lymphotropic virus type 1 (HTLV-1)-infected patients with HTLV-1-associated myelopathy/tropical spastic paraparesis, we cloned CTL from the peripheral blood and cerebrospinal fluid from patients with neurologic diseases and demonstrated the presence of HLA-A2, A3, and B14 restricted responses to the HTLV-1 p40x (tax) protein. We identified the minimal amino acid residues within the epitopes required for binding and recognition by HLA-A2- and B14-restricted CTL, identified the critical residues within the peptide sequence defining the HLA-A2-restricted response, and demonstrated that CTL can lyse T cells infected with HTLV-1. This study shows that the CTL response to HTLV-1 tax in patients with neurologic diseases is heterogenous in nature and is not confined to patients of a single HLA haplotype or to a specific region of the tax protein.

Identification and characterization of a novel family of pneumococcal proteins that are protective against sepsis.

Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

Role of lipopolysaccharide phase variation in susceptibility of Haemophilus influenzae to bactericidal immunoglobulin M antibodies in rabbit sera.

The effect of phase variation of lipopolysaccharide (LPS) structure on the susceptibility of Haemophilus influenzae to complement-dependent killing by normal human sera and normal rat sera has been described previously. The phase-variable structure phosphorylcholine (ChoP) confers susceptibility to human serum, since ChoP on the bacterial cell surface binds to serum C-reactive protein and activates complement. In contrast, expression of galalpha1,4gal, a second phase-variable epitope that is also found on human glycoconjugates, confers resistance to human serum. We studied the role of phase variation of these structures in the susceptibilities of H. influenzae KW20 (Rd) and a clinical isolate of nontypeable H. influenzae to killing by rabbit sera, which often possess naturally acquired complement-dependent bactericidal activity for unencapsulated H. influenzae. Expression of ChoP increased the resistance of strain KW20 to killing by bactericidal rabbit sera. In contrast, the serum resistance of a clinical isolate, H233, was unaffected by ChoP expression but was reduced by galalpha1,4gal expression. The rabbit sera with bactericidal activity (but not the nonbactericidal sera) all contained immunoglobulin M (IgM) antibodies able to bind to the surface of H. influenzae bacteria, as detected by flow cytometry, and contained IgM antibodies to LPS purified from strain KW20. Preincubation of sera with LPS reduced their bactericidal activity. Bactericidal activity was recovered quantitatively in an IgM-enriched fraction of sera. It is concluded that naturally occurring bactericidal activity for unencapsulated H. influenzae is largely due to IgM antibodies directed against phase-variable structures of the LPS.

Use of a whole genome approach to identify vaccine molecules affording protection against Streptococcus pneumoniae infection.

Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.