NFKBIZ polymorphisms and susceptibility to pneumococcal disease in European and African populations.

The proinflammatory transcription factor nuclear factor-kappaB (NF-kappaB) has a central role in host defence against pneumococcal disease. Both rare mutations and common polymorphisms in the NFKBIA gene encoding the NF-kappaB inhibitor, IkappaB-alpha, associate with susceptibility to bacterial disease, but the possible role of polymorphisms within the related IkappaB-zeta gene NFKBIZ in the development of invasive pneumococcal disease (IPD) has not been reported previously. To investigate this further, we examined the frequencies of 22 single-nucleotide polymorphisms spanning NFKBIZ in two case-control studies, comprising UK Caucasian (n=1008) and Kenyan (n=723) individuals. Nine polymorphisms within a single UK linkage disequilibrium (LD) block and all four polymorphisms within the equivalent, shorter Kenyan LD block displayed either a significant association with IPD or a trend towards association. For each polymorphism, heterozygosity was associated with protection from IPD when compared with the combined homozygous states (for example, for rs600718, Mantel-Haenszel 2 x 2 chi(2)=7.576, P=0.006, odds ratio (OR)=0.67, 95% confidence interval (95% CI) for OR: 0.51-0.88; for rs616597, Mantel-Haenszel 2 x 2 chi(2)=8.715, P=0.003, OR=0.65, 95% CI: 0.49-0.86). We conclude that multiple NFKBIZ polymorphisms associate with susceptibility to IPD in humans. The study of multiple populations may aid in fine mapping of associations within extensive regions of strong LD ('transethnic mapping').

Positive replication and linkage disequilibrium mapping of the chromosome 21q22.1 malaria susceptibility locus.

Four cytokine receptor genes are located on Chr21q22.11, encoding the alpha and beta subunits of the interferon-alpha receptor (IFNAR1 and IFNAR2), the beta subunit of the interleukin 10 receptor (IL10RB) and the second subunit of the interferon-gamma receptor (IFNGR2). We previously reported that two variants in IFNAR1 were associated with susceptibility to malaria in Gambians. We now present an extensive fine-scale mapping of the associated region utilizing 45 additional genetic markers obtained from public databases and by sequencing a 44 kb region in and around the IFNAR1 gene in 24 Gambian children (12 cases/12 controls). Within the IFNAR1 gene, a newly studied C --> G single-nucleotide polymorphism (IFNAR1 272354c-g) at position -576 relative to the transcription start was found to be more strongly associated with susceptibility to severe malaria. Association was observed in three populations: in Gambian (P=0.002), Kenyan (P=0.022) and Vietnamese (P=0.005) case-control studies. When all three studies were combined, using the Mantel-Haenszel test, the presence of IFNAR1 -576G was associated with a substantially elevated risk of severe malaria (N=2444, OR=1.38, 95% CI: 1.17-1.64; P=1.7 x 10(-4)). This study builds on previous work to further highlight the importance of the type-I interferon pathway in malaria susceptibility and illustrates the utility of typing SNPs within regions of high linkage disequilibrium in multiple populations to confirm initial positive associations.

Human immunodeficiency virus type 1 subtype C molecular phylogeny: consensus sequence for an AIDS vaccine design?

An evolving dominance of human immunodeficiency virus type 1 subtype C (HIV-1C) in the AIDS epidemic has been associated with a high prevalence of HIV-1C infection in the southern African countries and with an expanding epidemic in India and China. Understanding the molecular phylogeny and genetic diversity of HIV-1C viruses may be important for the design and evaluation of an HIV vaccine for ultimate use in the developing world. In this study we analyzed the phylogenetic relationships (i) between 73 non-recombinant HIV-1C near-full-length genome sequences, including 51 isolates from Botswana; (ii) between HIV-1C consensus sequences that represent different geographic subsets; and (iii) between specific isolates and consensus sequences. Based on the phylogenetic analyses of 73 near-full-length genomes, 16 "lineages" (a term that is used hereafter for discussion purposes and does not imply taxonomic standing) were identified within HIV-1C. The lineages were supported by high bootstrap values in maximum-parsimony and neighbor-joining analyses and were confirmed by the maximum-likelihood method. The nucleotide diversity between the 73 HIV-1C isolates (mean value of 8.93%; range, 2.9 to 11.7%) was significantly higher than the diversity of the samples to the consensus sequence (mean value of 4.86%; range, 3.3 to 7.2%, P < 0.0001). The translated amino acid distances to the consensus sequence were significantly lower than distances between samples within all HIV-1C proteins. The consensus sequences of HIV-1C proteins accompanied by amino acid frequencies were presented (that of Gag is presented in this work; those of Pol, Vif, Vpr, Tat, Rev, Vpu, Env, and Nef are presented elsewhere [http://www.aids.harvard.edu/lab_research/concensus_sequence.htm]). Additionally, in the promoter region three NF-kappa B sites (GGGRNNYYCC) were identified within the consensus sequences of the entire set or any subset of HIV-1C isolates. This study suggests that the consensus sequence approach could overcome the high genetic diversity of HIV-1C and facilitate an AIDS vaccine design, particularly if the assumption that an HIV-1C antigen with a more extensive match to the circulating viruses is likely to be more efficacious is proven in efficacy trials.

Identification of human immunodeficiency virus type 1 subtype C Gag-, Tat-, Rev-, and Nef-specific elispot-based cytotoxic T-lymphocyte responses for AIDS vaccine design.

The most severe human immunodeficiency virus type 1 (HIV-1) epidemic is occurring in southern Africa. It is caused by HIV-1 subtype C (HIV-1C). In this study we present the identification and analysis of cumulative cytotoxic T-lymphocyte (CTL) responses in the southern African country of Botswana. CTLs were shown to be an important component of the immune response to control HIV-1 infection. The definition of optimal and dominant epitopes across the HIV-1C genome that are targeted by CTL is critical for vaccine design. The characteristics of the predominant virus that causes the HIV-1 epidemic in a certain geographic area and also the genetic background of the population, through the distribution of common HLA class I alleles, might impact dominant CTL responses in the vaccinee and in the general population. The enzyme-linked immunospot (Elispot) gamma interferon assay has recently been shown to be a reliable tool to map optimal CTL epitopes, correlating well with other methods, such as intracellular staining, tetramer staining, and the classical chromium release assay. Using Elispot with overlapping synthetic peptides across Gag, Tat, Rev, and Nef, we analyzed HIV-1C-specific CTL responses of HIV-1-infected blood donors. Profiles of cumulative Elispot-based CTL responses combined with diversity and sequence consensus data provide an additional characterization of immunodominant regions across the HIV-1C genome. Results of the study suggest that the construction of a poly-epitope subtype-specific HIV-1 vaccine that includes multiple copies of immunodominant CTL epitopes across the viral genome, derived from predominant HIV-1 viruses, might be a logical approach to the design of a vaccine against AIDS.

The molecular epidemiology of HIV type 1 of men in Mexico.

Genotypic characteristics of human immunodeficiency virus type 1 (HIV-1) in Mexico were investigated in a multicenter study that involved centers in five geographic regions of the country. Study samples (n = 65) collected from male patients in 1998-1999 were sequenced within the C2-V5 region of the gp120 env gene. Phylogenetic analysis revealed that subtype B predominates in Mexico. The level of interpatient nucleotide diversity (mean value of 8.9%) was congruent with multiple introductions of the virus and the "aging" epidemic in Mexico. One-third of samples (30.8% of cases) showed polymorphism within the crown of the V3 loop demonstrating non-GPGR motifs. Two new motifs in the V3 loop crown - HPGG and GPEG - were observed. The evolution of the AIDS epidemic in Mexico should be closely monitored since non-B HIV-1 subtypes might be introduced. The nucleotide sequences were deposited in the GenBank under accession numbers AF200855-AF200869, AF200871-AF200892, and AF200894-AF200921.

HIV type 1 A/J recombinant with a pronounced pol gene mosaicism.

A nearly full-length genome sequence of a novel HIV-1 A/J recombinant with a complex structure of the pol gene has been analyzed. This virus was isolated in 1998 from a 35-year-old female from Botswana. The virus demonstrated a dual pattern for CXCR4/CCR5 coreceptor utilization. Using short-term enrichment of the donor's PBMCs, the 98BW21 isolate was long-range amplified, cloned, and sequenced. The sequence of the clone 98BW21.17 spanned 9103 bp from the PBS site to the U5 region of the 3' LTR. The phylogenetic relationship of the 98BW21.17 clone to HIV-1 sequences represented by M, N, and O groups and A-K subtypes of the M group was examined across the entire viral genome. The 98BW21.17 clone demonstrated a unique phylogenetic topology clustering within subtype A or subtype J reference sequences. However, the subtype origin of two regions within the pol gene (p51 RT and integrase) could not be identified. Recombination patterns of the 98BW21.17 clone were different from known AGJ/AGIJ-type viruses such as isolates BFP90 and 95ML84. This study demonstrated the existence and replication competence of a new dual-tropic X4/R5 recombinant form of HIV-1 on the subtype J backbone. The nucleotide sequence of the 98BW21.17 clone was submitted to GenBank under accession number AF192135.

Emerging recombinant human immunodeficiency viruses: uneven representation of the envelope V3 region.

OBJECTIVE: To determine whether the envelope V3 region from HIV-1 subtypes A, C or D had the same probability of being present in intersubtype recombinant genomes. MATERIALS AND METHODS: The envelope C2-C5 and the gag p24-p7 regions from one hundred infants infected perinatally in Tanzania were compared using phylogenetic and recombination analysis. Exact binomial and Fisher's exact tests were used to assess if various genomic regions were more likely to be overrepresented in intersubtype recombinants. RESULTS: Of one hundred HIV-1 positive infants analyzed, twenty-two (22%) showed exclusively subtype A sequence in gag and env. Subtype C accounted for twenty-two infants (22%) whereas nineteen infants (19%) were infected by HIV-1 subtype D. Intersubtype recombinant genomes accounted for thirty-seven infections (37%). The V3 region from subtype A was found in all fifteen A-D recombinants (P = 0.00003) and the V3 region from subtype C was found in all twelve C-D recombinants (P = 0.0002). Conversely, subtype D gag sequences were preferentially represented in the gag of A-D recombinants (P = 0.0003) as well as C-D recombinants (P = 0.002). In A-D recombinants, the V3 region of subtype A was generally surrounded by subtype A C3-C5 sequences. In contrast, the V3 region from subtype C was surrounded by subtype D C3-C5 sequences in C-D recombinants. Significant differences were not found in the number of subtype A or subtype C sequences in A-C recombinants. CONCLUSION: We have shown that several recombinant HIV-1 viruses have been generated and efficiently transmitted to infants in Tanzania. The recombination patterns showed that the V3 region of subtypes A or C was always selected in A-D and C-D recombinants. This selection suggests that the fitness of subtype D-V3 in perinatal transmission may be reduced with respect to V3 from subtype A and/or subtype C. The elevated number of recombinants transmitted perinatally suggests that co-infection or super-infection by two HIV-1 subtypes is not uncommon in this population.

Molecular cloning and phylogenetic analysis of human immunodeficiency virus type 1 subtype C: a set of 23 full-length clones from Botswana.

To better understand the virological aspect of the expanding AIDS epidemic in southern Africa, a set of 23 near-full-length clones of human immunodeficiency virus type 1 (HIV-1) representing eight AIDS patients from Botswana were sequenced and analyzed phylogenetically. All study viruses from Botswana belonged to HIV-1 subtype C. The interpatient diversity of the clones from Botswana was higher than among full-length isolates of subtype B or among a set of full-length HIV-1 genomes of subtype C from India (mean value of 9. 1% versus 6.5 and 4.3%, respectively; P < 0.0001 for both comparisons). Similar results were observed in all genes across the entire viral genome. We suggest that the high level of HIV-1 diversity might be a typical feature of the subtype C epidemic in southern Africa. The reason or reasons for this diversity are unclear, but may include an altered replication efficiency of HIV-1 subtype C and/or the multiple introduction of different subtype C viruses.

Identification of genes differentially expressed in Mycobacterium tuberculosis by differential display PCR.

RNA arbitrarily-primed differential display PCR (RAP-PCR) was used to identify and isolate genes differentially expressed between attenuated (H37Ra) and virulent (H37Rv, Erdman) laboratory strains of Mycobacterium tuberculosis (Mtb). Using this method, cDNA fragments showing homology to three known mycobacterial genes and six putative novel genes in mycobacterial cosmid vectors were identified. Among the putative novel Mtb genes identified, we found: (1) gene MTV041.29, containing multiple tandem repetitive sequences and encoding a putative Gly-, Ala, Asn-rich protein (PPE family); (2) gene MTV004.03, containing the AT10S repetitive gene sequence; (3) gene MTV028.09, encoding a hypothetical protein of unknown function; (4) genes MTCY78.20,21, possibly encoding two hypothetical proteins of unknown function; (5) gene MTCY01A6.09, encoding a putative novel ferrodoxin dependent glutamate synthase; and (6) gene MTCY31.20, encoding a putative cyclohexanone monooxygenase. Using gene specific primers in a second differential display PCR and by RT-PCR amplification, novel genes 1, 2, 3 and 4 were shown to be differentially up-regulated in the attenuated Mtb strain H37Ra compared to H37Rv and Erdman strain. Overall, we demonstrated that RAP-PCR, as a first step, is a quick and sensitive method for the identification and isolation of novel genes expressed in Mtb. Because of limitations inherent to the lack of specificity of arbitrary primers in the RAP-PCR method, a second differential display PCR and RT-PCR amplification with gene-specific primers was necessary in order to confirm differential expression of the identified genes.

Detailed analysis of a 17q21 microdissection library by sequence bioinformatics and isolation of region-specific clones.

A region-specific microdissection library originating from human chromosome 17q21, was constructed using the MboI linker-adaptor microcloning technique. DNA sequencing of 241 microclones resulted in the identification of 74 novel coding sequences, paralogs of known genes, and known, but previously unmapped, genes or expressed sequence tags that were "virtually" mapped to chromosome 17q21. By pooling the microclones as multiplexed hybridization probes, and by virtue of their origin on 17q21, we were able to identify approximately 150 P1 clones from the human Reference Library Data Base P1 Library that potentially map to chromosome 17q21. Verification of the 17q21 location of 16 P1 clones was accomplished by PCR analysis with STS primer pairs to known 17q21 genes or by FISH. Our results demonstrate the substantial advantage of combining the sequence analysis of microclones with multiplex hybridization strategies for gene discovery and mapping specific gene rich regions of the genome.