Cervical human papillomavirus infection and squamous intraepithelial lesions in rural Gambia, West Africa: viral sequence analysis and epidemiology.

The development of effective strategies against cervical cancer in Africa requires accurate type specific data on human papillomavirus (HPV) prevalence, including determination of DNA sequences in order to maximise local vaccine efficacy. We have investigated cervical HPV infection and squamous intraepithelial lesions (SIL) in an unselected cohort of 1061 women in a rural Gambian community. Squamous intraepithelial lesions was diagnosed using cytology and histology, HPV was typed by PCR-ELISA of DNA extracts, which were also DNA sequenced. The prevalence of cervical HPV infection was 13% and SIL were observed in 7% of subjects. Human papillomavirus-16 was most prevalent and most strongly associated with SIL. Also common were HPV-18, -33, -58 and, notably, -35. Human papillomavirus DNA sequencing revealed HPV-16 samples to be exclusively African type 1 (Af1). Subjects of the Wolof ethnic group had a lower prevalence of HPV infection while subjects aged 25-44 years had a higher prevalence of cervical precancer than older or younger subjects. This first report of HPV prevalence in an unselected, unscreened rural population confirms high rates of SIL and HPV infection in West Africa. This study has implications for the vaccination of Gambian and other African populations in the prevention of cervical cancer.

T cell responses to vaccines in infants: defective IFNgamma production after oral polio vaccination.

The immaturity of the neonatal immune system is associated with an increased susceptibility to infections. Studies in mice indicate that neonatal immune responses are biased towards the T helper 2 type, but little is known about helper T cell responses in human newborns. In this study, the oral polio vaccine was used as a model of early immunization to investigate the capacity of young infants to develop cellular immune responses. We show that neonatal immunization with oral polio vaccine induces the production of high titres of neutralizing antibodies but reduced proliferative and IFNgamma responses to polio antigens compared to immune adults. These data suggest that specific strategies will be required to immunize newborns against pathogens controlled by Th1 type immune responses.

Requirement for CD28 co-stimulation is lower in SHP-1-deficient T cells.

This study provides biochemical and functional evidence pertaining to the role of the intracellular protein tyrosine phosphatase, SHP-1, in influencing thresholds for TCR activation. Although the loss of SHP-1 in thymocytes from motheaten mice had minimal effects on the initial rise of cytosolic Ca(2+) concentration following TCR triggering, the post-stimulation equilibrium levels of Ca(2+) were consistently elevated. In keeping with a SHP-1 effect on PLCgamma function, IP3 generation was increased in SHP-1 deficient thymocytes. Importantly, we demonstrate that loss of SHP-1 results in a relaxation of the normally stringent co-stimulatory requirements for IL-2 production. SHP-1 deficient single-positive CD4(+) thymocytes revealed a significantly enhanced capacity to produce IL-2 in response to anti-CD3 stimulation alone. In contrast, the simultaneous triggering of CD3 and CD28 was required for equivalent IL-2 production in control single-positive CD4(+) thymocytes. Furthermore, SHP-1 deficient thymocytes generated an increased and prolonged proliferative response to anti-CD3 stimulation alone. In addition, the simultaneous triggering of CD28 and CD3 resulted in equivalent proliferative responses in SHP-1-deficient and control thymocytes, suggesting that a strong co-stimulatory signal is able to override the effect of SHP-1 loss on TCR hyperresponsiveness. Collectively, these results suggest that SHP-1, rather than acting directly on TCR signaling, may indirectly raise thresholds for TCR triggering by modulating co-stimulatory signals.

Influenza A antigen exposure selects dominant Vbeta17+ TCR in human CD8+ cytotoxic T cell responses.

During acute human viral infections, such as influenza A, specific cytotoxic T lymphocytes (CTL) are generated which aid virus clearance. We have observed that in HLA-A*0201+ subjects, CTL expressing Vbeta17+ TCR and recognizing a peptide from the influenza A matrix protein (M1(58-66)) dominate this response. In experimental models of infection such dominance can be due to inheritance of a restricted T cell repertoire or acquired consequent on expansion of CTL bearing an optimum TCR conformation against the MHC-peptide complex. To examine how influenza A infection might influence the development of TCR Vbeta17 expansion, we studied influenza A-specific CTL in a cross-sectional study of 82 HLA-A*0201+ individuals from birth (cord blood) to adulthood. Primary M1(58-66) -specific CTL were detected in cord blood, but their TCR were diverse and depletion of Vbeta17+ cells did not abrogate specific cytotoxicity. In contrast following natural influenza A infection, TCR Vbeta17+ CTL dominated to the extent that only one of nine adult CTL lines retained any functional activity after in vitro depletion of Vbeta17+ CTL. These results suggest that the dominance of Vbeta17+ TCR among adult M1(58-66)-specific CTL results from maturation and focussing of the response driven by exposure to influenza, and have implications for optimum immunization strategies.

Functional differences between influenza A-specific cytotoxic T lymphocyte clones expressing dominant and subdominant TCR.

We have shown that the dominance of CD8+ T cells expressing TCR Vbeta17 in the adult HLA-A*0201-restricted influenza A/M1(58-66)-specific response is acquired following first antigen exposure. Despite the acquired dominance of Vbeta17+ cells, subdominant M1(58-66)-specific clones expressing non-Vbeta17+ TCR persist in all individuals. To determine whether the affinity of the expressed TCR for the HLA-A*0201/M1(58-66) complex could influence functional properties, M1(58-66)-specific clones expressing subdominant (non-Vbeta17+) TCR were compared to cytotoxic T lymphocyte (CTL) clones expressing dominant (Vbeta17+) TCR. The Vbeta17+ CTL required up to 10,000-fold lower amounts of M1 peptide to mediate lysis compared to CTL clones expressing other Vbeta gene segments. All Vbeta17+ CTL clones tested bound HLA-A*0201/M1(58-66) tetramer, but two of three CTL clones expressing other TCR did not bind tetramer. The inability of non-Vbeta17+ CTL to bind tetramer did not correlate with phenotype, CD8 dependence or with cytokine production profiles. This suggests a limitation for the use of tetramers in examining subdominant T cell responses. Together these findings suggest that Vbeta17+ CTL which dominate the HLA-A*0201-restricted CTL response against influenza A are not functionally distinct from subdominant non-Vbeta17+ CTL. The dominance of Vbeta17+ CTL is likely to result from a competitive advantage due to superior CTL avidity for the HLA-A*0201/M1(58-66) complex.

Antigen processing defects in cervical carcinomas limit the presentation of a CTL epitope from human papillomavirus 16 E6.

Human papillomavirus (HPV) infection, particularly type 16, is causally associated with the development of cervical cancer. The E6 and E7 proteins of HPV are constitutively expressed in cervical carcinoma cells making them attractive targets for CTL-based immunotherapy. However, few studies have addressed whether cervical carcinomas can process and present HPV E6/E7-derived Ags for recognition by CTL. We generated HLA-A*0201-restricted CTL clones against HPV16 E6(29-38) that recognized HPV16 E6 Ags transfected into B lymphoblastoid cells. These CTL were unable to recognize HLA-A*0201(+) HPV16 E6(+) cervical carcinoma cell lines even when the level of endogenous HPV16 E6 in these cells was increased by transfection. This defect in presentation of HPV16 E6(29-38) correlated with low level expression of HLA class I, proteasome subunits low molecular mass protein 2 and 7, and the transporter proteins TAP1 and TAP2 in the cervical carcinoma cell lines. The expression of all of these proteins could be up-regulated by IFN-gamma, but this was insufficient for CTL recognition unless the level of HPV16 E6 Ag was also increased by transfection. CTL recognition of the HPV16 E6(29-38) epitope in 721.174 B cells was dependent on TAP expression but independent of immunoproteasome expression. Collectively, these findings suggest that presentation of the HPV16 E6(29-38) epitope in cervical carcinoma cell lines is limited both by the level of TAP expression and by the low level or availability of the source HPV E6 oncoprotein. These observations place constraints on the use of this, and potentially other, HPV-derived CTL epitopes for the immunotherapy of cervical cancer.

Novel method for detection, typing, and quantification of human papillomaviruses in clinical samples.

We report the development of a novel detection and typing methodology for human papillomaviruses (HPV) based on real-time PCR with the self-probing fluorescent primers known as Scorpions. This technique is quick, simple, specific, sensitive, and capable of estimating viral load per cell. We report the results of over 100 typing reactions performed on cell lines, biopsies, and cervical cytobrush samples which, when compared to the current reference HPV detection and typing technique, present a kappa value of 0.89. We further report preliminary data suggesting a relationship between viral load per cell and grade of cervical disease.

Human cytomegalovirus pp65- and immediate early 1 antigen-specific HLA class I-restricted cytotoxic T cell responses induced by cross-presentation of viral antigens.

Dendritic cells (DCs) play a pivotal role in the development of anti-viral CD8(+) CTL responses. This is straightforward if they are directly infected with virus, but is less clear in response to viruses that cannot productively infect DCS: Human CMV (HCMV) shows strain-specific cell tropism: fibroblast (Fb)-adapted laboratory strains (AD169) and recent clinical isolates do not infect DCs, whereas endothelial cell-adapted strains (TB40/E) result in productive lytic DC infection. However, we show here that uninfected DCs induce CD8(+) T cell cytotoxicity and IFN-gamma production against HCMV pp65 and immediate early 1 Ags following in vitro coculture with HCMV-AD169-infected Fbs, regardless of the HLA type of these FBS: CD8(+) T cell stimulation was inhibited by pretreatment of DCs with cytochalasin B or brefeldin A, indicating a phagosome/endosome to cytosol pathway. HCMV-infected Fbs were not apoptotic as measured by annexin V binding, and induction of apoptosis of infected Fbs in vitro did not augment CTL induction by DCs, suggesting a mechanism other than apoptosis in the initiation of cross-presentation. Furthermore, HCMV-infected Fbs provided a maturation signal for immature DCs during coculture, as evidenced by increased CD83 and HLA class II expression. Cross-presentation of HCMV Ags by host DCs enables these professional APCs to bypass some of the evasion mechanisms HCMV has developed to avoid T cell recognition. It may also serve to explain the presence of immediate early 1 Ag-specific CTLs in the face of pp65-induced inhibition of Ag presentation at the level of the infected cell.

Human papillomavirus type 16 E6/E7-specific cytotoxic T lymphocytes in women with cervical neoplasia.

Infection with oncogenic human papillomavirus (HPV) types is associated with the development of cervical neoplasia (CIN). The E6 and E7 oncoproteins are constitutively expressed in these lesions and are therefore putative targets for the immune response against HPV. The relation between HPV 16-specific memory cytotoxic T-cell precursor (mCTLp) activity to both oncoproteins and the natural course of cervical dysplasia was analyzed in 38 patients participating in a nonintervention cohort study of women with CIN and 11 HPV 16-positive cervical carcinoma patients. In a cross-sectional study at the end of follow-up prior to biopsy, 8 of 20 patients with a persistent HPV 16 infection had specific mCTLp against at least one of the two oncoproteins. By contrast, no specific mCTLp activity was detected in 11 HPV-negative patients or in 7 patients who had cleared an HPV 16 infection at the end of follow-up. However, 5 of 11 cervical carcinoma patients showed mCTLp activity against the E7 protein only. This study demonstrates that HPV 16 oncogene-specific mCTLp are present in women with HPV 16-positive CIN prior to any intervention. Since HPV-specific mCTLp were detected predominantly in women with high-grade lesions or invasive cervical carcinoma and not in women who cleared the virus, the role of naturally occurring mCTLp in the protection against HPV-associated cervical neoplasia remains to be established.

Innate recognition of bacteria in human milk is mediated by a milk-derived highly expressed pattern recognition receptor, soluble CD14.

Little is known about innate immunity to bacteria after birth in the hitherto sterile fetal intestine. Breast-feeding has long been associated with a lower incidence of gastrointestinal infections and inflammatory and allergic diseases. We found in human breast milk a 48-kD polypeptide, which we confirmed by mass spectrometry and sequencing to be a soluble form of the bacterial pattern recognition receptor CD14 (sCD14). Milk sCD14 (m-sCD14) concentrations were up to 20-fold higher than serum sCD14 from nonpregnant, pregnant, or lactating women. In contrast, lipopolysaccharide (LPS)-binding protein was at very low levels. Mammary epithelial cells produced 48-kD sCD14. m-sCD14 mediated activation by LPS and whole bacteria of CD14 negative cells, including intestinal epithelial cells, resulting in release of innate immune response molecules. m-sCD14 was undetectable in the infant formulas and commercial (cows') milk tested, although it was present in bovine colostrum. These findings indicate a sentinel role for sCD14 in human milk during bacterial colonization of the gut, and suggest that m-sCD14 may be involved in modulating local innate and adaptive immune responses, thus controlling homeostasis in the neonatal intestine.