Acting locally: innate mucosal immunity in resistance to HIV-1 infection in Kenyan commercial sex workers.

Cohort studies of female commercial sex workers (CSWs) in Kenya were among the first to identify highly HIV-1-exposed seronegative (HESN) individuals. As natural resistance is usually mediated by innate immune mechanisms, we focused on determining whether expression and function of innate signaling pathways were altered locally in the genital mucosa of HESN CSWs. Our results demonstrated that selected pattern-recognition receptors (PRRs) were significantly reduced in expression in cervical mononuclear cells (CMCs) from HESN compared with the new HIV-negative (HIV-N) and HIV-positive (HIV-P) groups. Although baseline levels of secreted cytokines were reduced in CMCs of HESN, they were highly stimulated following exposure to ssRNA40 in vitro. Importantly, cervical epithelial cells from HESN also expressed reduced levels of PRRs, but Toll-like receptor 3 (TLR3) and TLR7 as well as nuclear factor-κB and activator protein 1 were highly expressed and activated. Lastly, inflammatory cytokines interleukin (IL)-1ß, IL-8, and RANTES (regulated and normal T cell expressed and secreted) were detected at lower levels in cervicovaginal lavage of HESN compared with the HIV-N and HIV-P groups. Overall, our study reveals a local microenvironment of HIV resistance in the genital mucosa consisting of a finely controlled balance of basal immune quiescence with a focused and potent innate anti-viral response critical to resistance to sexual transmission of HIV-1.

Identification of three novel human leukocyte antigen alleles, HLA-B*58:43, HLA-C*03:190, and HLA-DPA1*01:12, in an East African cohort.

Three novel human leukocyte antigen (HLA) alleles were identified using a sequence-based typing of HLA class I and class II alleles of 1867 participants from a male circumcision cohort in Kenya. The new alleles were first identified by sequencing and then confirmed by cloning the polymerase chain reaction (PCR) products and sequencing multiple clones. HLA-B*58:43 was identical to HLA-B*58:02 with the exception of a nucleotide change at codon 125 in exon 3 (GCC→ACC), and resulted in the amino acid change from Alanine to Threonine. HLA-C*03:190 was identical to HLA-C*03:02:01 with the exception of a nucleotide change at codon 131 in exon 3 (CGC→TGC), and resulted in the amino acid change from Arginine to Cysteine. HLA-DPA1*01:12 was identical to HLA-DPA1*01:03:01:01 with the exception of a nucleotide change at codon 66 in exon 2 (TTG→TCG), and resulted in the amino acid change from Leucine to Serine.

HLA class I associations with rates of HIV-1 seroconversion and disease progression in the Pumwani Sex Worker Cohort.

Class I human leukocyte antigens (HLA) play an important role in the adaptive immune response by presenting antigens to CD8+ T cells. Studies have reported that several HLA class I alleles are associated with differential disease progression in human immunodeficiency virus (HIV)-infected individuals, however, few class I associations with resistance or susceptibility to HIV-1 infection have been reported. We typed HLA-A, -B and -C of >1000 women enrolled in the Pumwani Sex Worker Cohort using a sequence-based typing method. Kaplan-Meier analysis was used to identify alleles influencing seroconversion and disease progression to acquired immune deficiency syndrome (CD4 < 200/mm³). A*01 (P = 0.020), C*06:02 (P = 0.042) and C*07:01 (P = 0.050) are independently associated with protection from seroconversion. Women with any of these alleles are less likely to seroconvert [P = 0.00001, odds ratio (OR): 0.503, 95% confidence interval (CI): 0.320-0.790]. Conversely, A*23:01 (P = 0.004), B*07:02 (P = 0.003) and B*42:01 (P = 0.025) are independently associated with rapid seroconversion. Women with any of these alleles are twice as likely to seroconvert (P = 0.002, OR: 2.059, 95% CI: 1.290-3.285). The beneficial alleles confer threefold protection from seroconversion when compared with the susceptible alleles (P = 0.000001, OR: 0.268, 95% CI: 0.132-0.544). B*07:02 is the contributing allele, within the B7 supertype, to the rapid seroconversion. A*74:01 (P = 0.04/P = 0.006), B*14 (P = 0.003/P = 0.003) and B*57:03 (P = 0.012/P = 0.038) are independently associated with slower CD4+ decline and LTNP phenotype, while B*07:02 (P = 0.020), B*15:10 (P = 0.022) and B*53:01 (P = 0.007) are independently associated with rapid CD4+ T-cell decline. B7 supertype (P = 0.00006), B*35*-Py (P = 0.028) and B*35-Px (P = 0.001) were also significantly associated with rapid CD4+ T-cell decline. Understanding why these HLA class I alleles are associated with protection/susceptibility to HIV-1 acquisition and disease progression could contribute to the development of effective prophylactic and therapeutic vaccines for HIV-1.

Evidence of recombination producing allelic diversity in MHC class I Mafa-B and -A alleles in cynomolgus macaques.

The MHC class I-A and -B genes of cynomolgus macaques are highly polymorphic. These genes encode proteins presenting peptides to CD8+ T cells to initiate adaptive immune response. Recombination events are one way the diversity of these alleles can be increased. Such events have been well characterized in humans, but have not been as well characterized in macaques. In order to identify and examine recombinations that create new alleles, it is important to analyze intron sequences. Intron sequences have been shown to be important to understand the evolutionary mechanisms involved in the generation of major histocompatibility complex (MHC) alleles and loci. Thus far, there have been relatively few intron sequences reported for MHC class I alleles in macaques, and this has hampered the understanding of MHC organization and evolution in macaques. In this study, we present evidence of a gene conversion event generating the Mafa-B*099 allele lineage by the combination of Mafa-B*054 and Mafa-B*095 allele lineages. A potential recombination between the Mafa-A3*13 and Mafa-A4:14 lineages was also observed, but it is less clear due to lack of intron 2 sequence. This report stresses the role that recombination can play in MHC class I diversity in cynomologus macaques, and the importance of introns in identifying and analyzing such events.

A distinct cytokine and chemokine profile at the genital mucosa is associated with HIV-1 protection among HIV-exposed seronegative commercial sex workers.

The predominance of HIV-1 sexual transmission requires a greater understanding of the interaction between HIV-1 and the mucosal immune system. The study of HIV-1-exposed seronegative (HESN) individuals serves as a model to identify the correlates of protection and to aid in microbicide development. A total of 22 cytokines/chemokines were analyzed at the systemic and mucosal compartments in 57 HESN, 51 HIV-1-negative, and 67 HIV-1-infected commercial sex workers from Nairobi, Kenya. HESN individuals had significantly lower expression of monokine induced by interferon-γ (MIG), interferon-γ-induced protein 10 (IP-10), and interleukin-1α (IL-1α) in their genital mucosa compared with controls. HESN cytokine expression also distinctly correlates with mucosal antiproteases, suggesting that HESN individuals have a unique pattern of mucosal chemokine/cytokine expression, which may result in reduced trafficking at the mucosa. These data support the immune quiescence model of protection, whereby lower T-cell activation/recruitment at the mucosal compartment reduces HIV-1 target cell numbers and is an important component of natural protection from HIV-1.

Identification of 23 novel MHC class I alleles in cynomolgus macaques of Philippine and Philippine/Mauritius origins.

We report here novel Mafa-A, -AG and -B alleles identified in two groups of cynomolgus macaques.

Comprehensive proteomic study identifies serpin and cystatin antiproteases as novel correlates of HIV-1 resistance in the cervicovaginal mucosa of female sex workers.

Not all individuals exposed to HIV-1 become infected, and evidence from HIV-1 highly exposed seronegative women (HIV-1-resistant) suggests that mucosal factors in the female genital tract, the first site of contact for the virus, are playing a role. To better understand factors mediating protection from HIV-1, we performed a large clinical study using the tools of systems biology to fully characterize the cervicovaginal mucosa proteome in HIV-1-resistant women. Cervicovaginal lavage fluid was collected from 293 HIV-1-resistant, uninfected, and infected sex workers and analyzed by 2D-LC LTQ-FT-MS. Of the more than 360 unique proteins identified, 41 were differentially abundant (>3-fold cutoff) in HIV-1-resistant women. The majority of over-abundant proteins were antiproteases (>40%), some with described anti-inflammatory and anti-HIV-1 activity. Quantification of specific anti-HIV-1 antiproteases Serpin A1, Serpin A3, and Cystatin B and an epithelial antiprotease A2ML1 found them to be significantly over-abundant in HIV-1-resistant women (p = 0.004; p = 0.046; p = 0.0003; and p = 0.04, respectively). Expression levels were not correlated to sexual practices or other epidemiological factors. Mucosal antiprotease levels correlated with pro-inflammatory cytokine concentration (p = <0.0001), but independently of pro-inflammatory cytokine levels in HIV-1-resistant women including TNF-alpha, IL-1 alpha, IL-1 beta, IL-6, and IL-8. This comprehensive systems biology approach identifies mucosal serpins and cystatins as novel correlates of HIV-1-resistance. This represents the first study characterizing these factors in the female genital tract.

Identification of a novel HLA-A allele, A*29:28, in an East African population.

The new allele is identical to A*29:01:01:01 in exons 2 and 3, except for a single-nucleotide substitution (TTG to TGG) at codon 156.

Cellular immune responses to recurring influenza strains have limited boosting ability and limited cross-reactivity to other strains.

Influenza vaccine provides protection against infection with matched strains, and this protection correlates with serum antibody titres. In addition to antibodies, influenza-specific CD8+ T-lymphocyte responses are important in decreasing disease severity and facilitating viral clearance. Because this response is directed at internal, relatively conserved antigens, it affords some cross-protection within a given subtype of influenza virus. With the possibility of a broader A(H1N1) Mexico outbreak in the fall of 2009, it appeared worthwhile studying the degree of cellular immune response-mediated cross-reactivity among influenza virus isolates. The composition of the 2006-2007 influenza vaccine included the A/New Caledonia/20/1999 strain (comprising a virus that has been circulating, and was included in vaccine preparations, for 6-7 years) and two strains not previously included (Wisconsin and Malaysia). This combination afforded us the opportunity to determine the degree of cross-reactive cellular immunity after exposure to new viral strains. We analysed the antibody responses and the phenotype and function of the T cell response to vaccine components. The results obtained show that antibody responses to A/New-Caledonia were already high and vaccination did not increase antibody or cytotoxic T lymphocyte responses. These data suggest that repeated exposure to the same influenza stain results in limited boosting of humoral and cellular immune responses.

Systems biology-based approaches to understand HIV-exposed uninfected women.

Worldwide HIV infects women more frequently than men, and it is clear that not all exposed to HIV become infected. Several populations of HIV-exposed uninfected (EU) women have been identified, including discordant couples and sex workers. Understanding what provides natural protection in EU women is critical in vaccine or microbicide development. However, correlates of protection in these women are still unclear. Most studies have used classical methods, examining single genes or cellular factors, a mainstay for traditional immunobiology. This reductionist approach may be limited in the information it can provide. Novel technologies are now available that allow us to take a "systems biology" approach, which allows the study of a complex biological system and identifies factors that may provide protection against HIV infection. Herein we report developments in discovery-based systems biology approaches in EU women and how this broadens our understanding of natural protection against HIV-1.

Identification of four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, from an East African population by high-resolution sequence-based typing.

We report here four novel HLA-B alleles, B*1590, B*1591, B*2726, and B*4705, identified from an East African population during sequence-based HLA-B typing. The novel alleles were confirmed by sequencing two separate polymerase chain reaction products, and by molecular cloning and sequencing multiple clones. B*1590 is identical to B*1510 at exon 2 and exon 3, except for a difference (GCCGTC) at codon 158. Sequence differences at codon 152 (GAGGTG) and codon 167 (TGGTCG) differentiate B*1591 from B*1503 at exon 3. B*2726 is identical to B*2708 at exon 2 and exon 3, except for a difference (AAGCAG) at codon 70. B*4705 was identified in three Kenyan women. The allele is identical to B*47010101/02 at exon 2 and exon 3, except for differences at codon 97 (AGGAAT) and codon 99 (TTTTAT). These new alleles have been named by the WHO Nomenclature Committee. Identification of these novel HLA-B alleles reflects the genetic diversity of this East African population.

High-resolution sequence-based DPA1 typing identified two novel DPA1 alleles, DPA1*010303 and DPA1*0303, from a Kenyan population.

We report here two novel DPA1 alleles, DPA1*010303 and DPA1*0303, identified from a Kenyan population during sequence-based HLA-DPA1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPA1 alleles is identical to DPA1*010301 at exon 2, except for a single nucleotide substitution (ACG ACC) at codon 15. The new allele has been named by the WHO Nomenclature Committee as DPA1*010303. The second novel DPA1 allele is identical to DPA1*0301, except for a single nucleotide difference (GAA GAC) at codon 28 that changed the amino acid from Glu to Asp. The new allele has been named by the WHO Nomenclature Committee as DPA1*0303. Identification of the two novel DPA1 alleles reflects the genetic diversity of this East African population.

Identification of a novel HLA-DQA1 null allele, DQA1*0403N, from an East African woman.

We report a novel DQA1 allele (DQA1*0403N) identified during sequence-based HLA-DQA1 typing of a Kenyan population. The new allele is identical to DQA1*0401 at exon 2 except for a single-nucleotide substitution at codon 53, changing it from lysine to a stop codon (CAA-->TAA). The substitution at codon 53 was confirmed by sequencing two separate polymerase chain reaction products and by sequencing multiple clones obtained following TOPO-TA cloning. The resulting stop codon at position of codon 53 in exon 2 is predicted to produce a non-functional DQA1 alpha-chain. The new allele has been named by the WHO nomenclature committee as DQA1*0403N. This is the first report of a null allele detected in the DQA1 gene.

High resolution sequence-based DPB1 typing identified two novel DPB1 alleles, DPB1*9401 and DPB1*9501, from a Kenyan population.

Two novel DPB1 alleles, DPB1*9401 and DPB1*9501, were identified from a Kenyan population during sequence-based HLA-DPB1 typing. Molecular cloning and sequencing of multiple clones confirmed that one of the new DPB1 alleles is identical to DPB1*0402 at exon 2 except for a single nucleotide substitution (CGG -->TGG), changing codon 70 from Arg to Trp. The new allele has been named DPB1*9401. This is the first report of polymorphism at codon 70 of HLA-DPB1 alleles. New codon combinations have been identified in another novel DPB1 allele named DPB1*9501. The extensive diversity at DPB1 locus of this East African population is being revealed by high resolution sequence-based DPB1 typing.

Improved mRNA quantitation in LightCycler RT-PCR.

BACKGROUND: Real-time polymerase chain reaction (PCR) utilizing the LightCycler and similar systems is an increasingly used technique for quantitative reverse transcription (RT)-PCR of mRNA levels from genes of immunologic interest. A commonly encountered limitation with these systems is that the fluorescence induced by SYBR Green (a fluorophore that binds double-stranded DNA) can result from primer dimers (PDs) as well as the PCR product of interest, thus interfering with the ability to reproducibly quantitate mRNA levels. METHODS: We use a modification of the LightCycler PCR strategy to overcome this problem by altering the PCR strategy to take advantage of the LightCycler's ability to measure fluorescence at a temperature greater than the melting point of PDs. The resulting measurements determine fluorescence of only the desired PCR product. RESULTS: We demonstrate that by using this modified PCR strategy, one can eliminate the fluorescence induced by PDs and obtain accurate product quantitation. CONCLUSIONS: This simple modification allows more precise quantitation of sample mRNA levels by eliminating the contaminating fluorescence induced by the formation of PCR PDs. This modification obviates the need to redesign PCR primers in RT-PCR experiments where this is impractical or impossible.

HLA-A and HLA-B in Kenya, Africa: allele frequencies and identification of HLA-B*1567 and HLA-B*4426.

HLA-A and HLA-B alleles of a population from Kenya, Africa were examined by sequencing exon 2 and exon 3 DNA and typing using a Taxonomy-based Sequence-analysis (TBSA) method. Extensive diversities were observed at both HLA-A and HLA-B loci in this population. Forty-one HLA-A alleles were identified from 159 unrelated individuals. The most frequently observed alleles were A*6802 (11.64%), A*02011/09 (9.75%), A*7401/02 (9.43%), A*3001 (7.86%), A*3002 (7.23%) and A*3601 (6.6%). Forty-nine HLA-B alleles were identified in 161 unrelated individuals, including two novel alleles, B*1567 and B*4426. The most frequently observed HLA-B alleles were B*5301 (9.01%), B*5801 (8.38%), B*4201 (7.76%), B*1503 (7.14%), B*1801 (6.21%), and B*5802 (5.90%). The most frequently observed HLA-A-B haplotypes were A*3601-B*5301 (3.55%) and A*3001-B*4201 (3.19%), followed by A*7401/02-B*5801 (2.84%), A*7401/02-B*5802 (2.84%) and A*02011/09-B*1503 (2.13%). Linkage disequilibrium and chi2 analysis showed the association of these HLA-A-B haplotypes at the antigen level to be significant. The frequencies of HLA-A and HLA-B alleles from the Kenyan population were compared with that of a population from Cameroon. The difference in allele and haplotype frequency distributions partly reflected the different ethnic composition of these two African populations.

Response of a sexually transmitted infection epidemic to a treatment and prevention programme in Nairobi, Kenya.

Although it seems possible in a developing country context such as Kenya, given appropriate inputs and a sound approach, to shift a sexually transmitted disease (STI) epidemic from phase II to III, it is not entirely clear how to go beyond this stage, to low levels of endemicity or even elimination. Perhaps the most important challenge now is to expand STI treatment and community STI/HIV prevention programmes to a much larger scale. Although successful programmes have been implemented in many areas of sub-Saharan Africa on a small scale, a significant impact in reducing the STI/HIV burden will not occur until programme reach is expanded to district, provincial, and national levels.

How important is the 'quality' of the cytotoxic T lymphocyte (CTL) response in protection against HIV infection?

Cytotoxic T lymphocyte (CTL) responses have been associated with protection from HIV-1 infection in people with a high degree of exposure to HIV and who show no serological evidence of HIV infection (HEPS, highly exposed persistently seronegative). However, it remains unclear how protective CTL responses could apparently develop in a minority of people, whilst the great majority of HIV-infected people make strong CTL responses yet progress to AIDS and death. In this paper we review the data which supports the hypothesis that the quality of the T-cell response, rather than its magnitude, may be an important factor that merits further investigation.

New insights into HIV-1 specific cytotoxic T-lymphocyte responses in exposed, persistently seronegative Kenyan sex workers.

A clearer understanding of HIV-1 specific immune responses in highly-exposed, persistently seronegative (HEPS) subjects is important in developing models of HIV-1 protective immunity. HIV-1 specific cytotoxic T-lymphocytes (CTL) have been described in a cohort of HEPS Kenyan sex workers, and recent work has further elucidated these responses. CTL specific for HIV-1 Env were found in the blood of over half the sex workers meeting criteria for HIV resistance, and in some women recognized unmapped epitopes. The proportion of women with Env-specific CTL increased with the duration of uninfected HIV exposure, suggesting that these responses were acquired over time. CD8+ lymphocyte responses directed against predefined HIV-1 CTL epitopes from various HIV-1 genes were found in the blood and genital tract of >50% resistant sex workers, at a ten-fold lower frequency than in infected subjects. The epitope specificity of CD8+ responses differs between HEPS and HIV infected women, and in HEPS the maintenance of responses appears to be dependent on persistent HIV exposure. Several HIV-1 'resistant' sex workers have become HIV infected over the past 6 years, possibly related to waning of pre-existing HIV-specific CTL, and infection has often been associated with a switch in the epitope specificity of CD8+ responses. These findings suggest that vaccine-induced protective HIV immunity is a realistic goal, but that vaccine strategies of boosting or persistent antigen may be necessary for long-lived protection.

Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers.

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.