Genome Sequences of Three African Swine Fever Viruses of Genotypes IV and XX from Zaire and South Africa, Isolated from a Domestic Pig (Sus scrofa domesticus), a Warthog (Phacochoerus africanus), and a European Wild Boar (Sus scrofa).

We report here the genome sequences of three African swine fever virus isolates obtained from a domestic pig (Zaire [Zaire]), a warthog (RSA/W1/1999 [South Africa]), and a European wild boar (RSA/2/2004 [South Africa]) belonging to genotypes IV, XX, and XX, respectively. This report increases the number of genotype XX, wild boar, and warthog reference sequences available.

Genome Sequences of Three African Swine Fever Viruses of Genotypes I, III, and XXII from South Africa and Zambia, Isolated from Ornithodoros Soft Ticks.

Here, we report the draft genome sequences of three African swine fever viruses isolated from Ornithodoros soft ticks. Isolates LIV 5/40 (Zambia), SPEC 57 (South Africa), and RSA/2/2008 (South Africa) belong to genotypes I, III, and XXII, respectively.

Human papillomavirus types in HIV seropositive men with penile warts in Johannesburg, South Africa.

Few data exist regarding the human papillomavirus (HPV) types in penile warts (PW) of HIV-infected men in Africa. Nurses collected penile swabs for HPV typing from 74 HIV-positive men with PW. HPV genotyping was performed using the Roche Linear Array Test. Analysis was performed on data relating to 74 samples. The mean age of the men was 36.0 years and 78.5% (51/65) were uncircumcised. Of the 73/74 validated results, all tested positive for HPV; 42.5% (31/73) and 32.9% (24/73) had HPV types 6 and 11, respectively. 84.9% of men tested positive for any oncogenic type: 20/73 (27.4%) were positive for type 16, 11/73 (15.1%) were positive for type 18 and 8/73 (11.0%) men had both types. Our study shows a high prevalence (68.5%) of HPV type 6 and/or 11 in this male population with PW. Given the poor availability of treatment, a quadrivalent vaccine for men may have significant benefit.

HPV genotypes in women with squamous intraepithelial lesions and normal cervixes participating in a community-based microbicide study in Pretoria, South Africa.

BACKGROUND: Little is known regarding the human papillomaviruses (HPV) genotypes prevalent in women in South Africa, a country with a high incidence of cervical cancer. OBJECTIVE: To determine the prevalence and HPV genotypes in women with squamous abnormalities and normal cervixes participating in a community-based microbicide study. STUDY DESIGN: A total of 159 cervical specimens, including 56 specimens from women with abnormal cytology (cases) and 103 randomly selected specimens from women with normal cytology (controls), were collected. HPV was detected by consensus PCR primers and HPV genotypes were determined by Roche Linear Array HPV genotyping assay. RESULTS: HPV genotypes were found in 91% of cases and 40% of controls (p<0.005). High-risk HPV was detected in all high-grade squamous intraepithelial lesions (HSILs), 69% of low-grade squamous intraepithelial lesions (LSILs), 57% of atypical squamous cells of undetermined significance (ASCUS), and 86% of ASCUS in which HSIL could not be excluded (ASCUS-H), and 73% of HPV positive controls. HPV-35 was the predominant genotype in HSILs; HPV-18 in ASCUS; HPV-58 in ASCUS-H and HPV-16 in LSILs and controls. CONCLUSION: High-risk HPV prevalence was high in both cases and controls. HPV genotype distribution in HSILs was different from that reported worldwide and from other studies in South Africa.

No relationship observed between human p53 codon-72 genotype and HPV-associated cervical cancer in a population group with a low arginine-72 allele frequency.

Infection with high-risk human papillomavirus (HR-HPV) is a necessary but not a sufficient event in the development of cervical cancer, as most infections regress without intervention. Thus, genetic host factors and cellular immune responses could be potential modifiers for the risk of developing cervical cancer. In particular, p53 is considered as the most critical tumour suppressor gene and is involved in regulating cell division. The polymorphism on p53, which encodes either a proline or an arginine amino acid residue at codon 72, has been reported as a possible risk factor for cervical disease. This polymorphism has been shown to differentially affect the efficiency of degradation of p53 protein mediated by HR-HPV E6 oncoprotein. Women with histologically proven cancer of the cervix (n = 111) and hospital-based controls (n = 143) were included in this study. The patients and controls were from the Western Cape Province in South Africa. Genotyping of the p53 polymorphism was conducted using polymerase chain reaction and restriction fragment-length polymorphism method. The distributions of the allelic frequencies were stratified in both patients and controls into two South African ethnic population groups. In this study, we observed no association between the distribution of p53 polymorphism and susceptibility to cervical cancer in the Western Cape Province populations (P = 0.466). However, the frequency of the Pro/Pro residue at codon 72 was increased in the South African population when compared to Caucasians, Indians and Portuguese population groups. Notably, as the distribution of the Pro/Pro at codon 72 of p53 gene was significantly different (P < 0.05) between the control groups of South Africa and other population groups. This result suggests that ethnic disparity may influence the levels of p53 produced.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization.

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means.

Rabbits immunised with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L2E7E2 genes induces regression of established papillomas.

We previously demonstrated in a cottontail rabbit papillomavirus (CRPV) challenge model that recombinant Bacille Calmette-Guerin (rBCG) could potentially be used as a prophylactic vaccine vehicle to deliver papillomavirus proteins. In this study we investigated whether regression of CRPV-induced papillomas could be achieved following immunisation of out-bred New Zealand White rabbits with rBCG expressing CRPVL2, CRPVE2, CRPVE7 or CRPVL2E7E2 proteins. Rabbits immunised with rBCG/CRPVL2E7E2 had papillomas that were largely suppressed and were significantly smaller compared to the rBCG negative control group (P

Plant-produced cottontail rabbit papillomavirus L1 protein protects against tumor challenge: a proof-of-concept study.

The native cottontail rabbit papillomavirus (CRPV) L1 capsid protein gene was expressed transgenically via Agrobacterium tumefaciens transformation and transiently via a tobacco mosaic virus (TMV) vector in Nicotiana spp. L1 protein was detected in concentrated plant extracts at concentrations up to 1.0 mg/kg in transgenic plants and up to 0.4 mg/kg in TMV-infected plants. The protein did not detectably assemble into viruslike particles; however, immunoelectron microscopy showed presumptive pentamer aggregates, and extracted protein reacted with conformation-specific and neutralizing monoclonal antibodies. Rabbits were injected with concentrated protein extract with Freund's incomplete adjuvant. All sera reacted with baculovirus-produced CRPV L1; however, they did not detectably neutralize infectivity in an in vitro assay. Vaccinated rabbits were, however, protected against wart development on subsequent challenge with live virus. This is the first evidence that a plant-derived papillomavirus vaccine is protective in an animal model and is a proof of concept for human papillomavirus vaccines produced in plants.

Immunisation with recombinant BCG expressing the cottontail rabbit papillomavirus (CRPV) L1 gene provides protection from CRPV challenge.

Recombinant Bacille Calmette-Guerin (rBCG) could potentially be the vaccine vehicle of choice to deliver foreign antigens from multiple pathogens. In this study we have used the cottontail rabbit papillomavirus (CRPV) rabbit model to provide a "proof of concept" that immunisation with rBCG expressing the CRPV major capsid protein, L1 (rBCG/CRPVL1), will protect outbred New Zealand White rabbits against CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were protected 5 weeks post-CRPV challenge. Rabbits immunised with rBCG/CRPVL1 (10(5) cfu/ml) had papillomas, which were smaller and took longer to appear than the control rabbits. None of the negative control rabbits vaccinated with rBCG expressing an irrelevant gene or PBS were protected from CRPV challenge. Sera from rabbits immunised with rBCG/CRPVL1 (10(7) cfu/ml) were able to neutralise 54.5% of CRPV at serum dilutions of 1:200. These results provide evidence that BCG could potentially be used as a vaccine delivery vehicle for human papillomavirus proteins as a possible prophylactic vaccine.

The immune response of the Chacma baboon to Bacille Calmette Guerin: development of a primate model for BCG-based vaccine research.

Mycobacterium bovis Bacilli Calmette-Guerin (BCG) is used increasingly as an efficient vector for expression of recombinant proteins to induce a strong cell-mediated immunity. Here, we tested the immune response of Chacma baboons to the Tokyo and Pasteur strains of BCG in order to obtain base-line information on the response of this primate to BCG. While a humoral immune response to BCG was detected only in some vaccinated baboons, a cellular immune response characterized by a PPD-specific delayed hypersensitivity response and BCG-specific IFN-gamma production from PBMC was a consistent finding. These responses were long-lived and could be detected beyond a year after a booster inoculation at 20 weeks. The results thus suggest that the Chacma baboon may be used as a non-human primate for the evaluation of recombinant BCG vaccines.

Strategies for the prevention of cervical cancer by human papillomavirus vaccination.

As cervical cancer is causally associated with 14 high-risk types of human papillomavirus (HPV), a successful HPV vaccine will have a major impact on this disease. Although some persistent HPV infections progress to cervical cancer, host immunity is generally able to clear most HPV infections. Both cell-mediated and antibody responses have been implicated in influencing the susceptibility, persistence or clearance of genital HPV infection. There have been two clinical trials that show that vaccines based on virus-like particles (VLPs) made from the major capsid protein, L1, are able to type specifically protect against cervical intra-epithelial neoplasia and infection. However, there is no evidence that even a mixed VLP vaccine will protect against types not included in the vaccine, and a major challenge that remains is how to engineer protection across a broader spectrum of viruses. Strategies for production of HPV vaccines using different vaccine vectors and different production systems are also reviewed.

Expression of Human papillomavirus type 16 major capsid protein in transgenic Nicotiana tabacum cv. Xanthi.

The production of vaccine antigens in plants is a safe and potentially very cost-effective alternative to traditional expression systems. We investigated the possibility of transgenic plant expression of the Human papillomavirus (HPV) type 16 L1 major capsid protein, with and without nuclear localisation signals, in Nicotiana tabacum cv. Xanthi plants. The genes were stably integrated into the N. tabacum genome, and both the expressed proteins were capable of assembling into capsomers and virus-like particles. The proteins in concentrated leaf extracts (L1(Tr)) were tested for antigenicity using a panel of characterised monoclonal antibodies (Mabs). Neutralising and conformation-specific Mabs (H16:V5 and H16:E70) were shown to bind to both types of the plant-produced particles. We estimated the L1(Tr) product yield to be 2-4 microg per kg of fresh leaf material. Rabbits immunised with small doses of plant-produced particles elicited a weak anti-HPV-16 L1 immune response. Our results support the feasibility of using transgenic plants for the production of HPV vaccines.

Limited Pap screening associated with reduced risk of cervical cancer in South Africa.

BACKGROUND: We investigated the effect of Pap smear screening on the incidence of invasive cancer of the cervix in the Western Cape, South Africa where screening is limited. METHODS: Data were derived from a case-control study of the association of hormonal contraceptives and invasive cervical cancer. Incident cases (n = 524) of invasive cervical cancer who presented at two tertiary hospitals and controls (n = 1540) series matched for age, race, and place of residence were interviewed. Information on a wide range of variables was collected including whether the women had previously had a Pap smear taken and the number and timing of smears. Odds ratios (OR) and 95% CI were calculated using multiple logistic regression. RESULTS: The OR of cervical cancer was reduced among women who had ever had a smear (OR = 0.3, 95% CI: 0.3-0.4). The OR declined with increasing number of smears to 0.2 for >/=>3 smears (trend P = 0.0003). Among women who had a smear <5 years previously the OR was 0.3, but even if the smear was taken >/=15 years previously the women remained at reduced risk (OR = 0.5). CONCLUSION: The data suggest that even limited Pap smear screening reduces the risk of cervical cancer. Should a screening programme be successfully implemented, the incidence of cervical cancer might be reduced by as much as 70%.

Hookworm cathepsin D aspartic proteases: contributing roles in the host-specific degradation of serum proteins and skin macromolecules.

Cathepsin D aspartic proteases of hookworms were recently implicated in the host-specific digestion of haemoglobin by adult parasites. Ac-APR-1 from the dog hookworm, Ancylostoma caninum and Na-APR-1 from the human hookworm, Necator americanus, were shown to be expressed in the infective larval stage (L3) as well as adult worms. We now show that both proteases degraded skin macromolecules and serum proteins, some of which were cleaved more readily from permissive definitive hosts as opposed to non-permissive hosts. Na-APR-1 degraded human collagens more efficiently than did Ac-APR-1, and Ac-APR-1 degraded canine serum albumin more efficiently than did Na-APR-1. On the other hand, both enzymes degraded human serum proteins (albumin and fibrinogen) with approximately equal efficiency under the conditions of our assays in vitro. Molecular models of these 2 orthologous, aspartic proteases showed that, despite having active site clefts with identical primary sequences, residues in the S3 pocket adopted different conformations, likely accounting for different substrate preferences reported previously. Antisera raised to both proteases partially inhibited (16-26%) migration of hookworm L3 through hamster skin in vitro, further implying a connective tissue invasive role for these enzymes in addition to digestion of serum and erythrocyte proteins for nutrition.

Oral antibodies to human papillomavirus type 16 in women with cervical neoplasia.

This study investigated the relationship between human papillomavirus type 16 (HPV-16) antibodies detected in oral fluid from women with cervical neoplasia, their HPV-16 antibody seroprevalence, and their cervical HPV-16 DNA presence. Cervical HPV-16 DNA was detected by polymerase chain reaction in 43.2% (35/81) of these women. The prevalence of IgG and IgA antibodies to HPV-16 virus-like particles (VLP-16) in oral fluid and was investigated by enzyme-linked immunosorbent assay. Anti-VLP-16 IgA antibodies were detected in oral fluid from 54.3% (44/81) of women with cervical neoplasia, compared with 8% (3/36) in controls (P = 0.000002). Anti-VLP-16 IgG was detected in oral fluid from 43.2.9% (25/72) and 13.3% (4/30; P = 0.029), respectively. Women who were HPV-16 DNA positive at their cervical lesion, displayed an oral fluid anti-VLP-16 IgA prevalence of 60.7% (17/28) and HPV-16 DNA negative women an oral fluid anti-VLP-16 IgA prevalence of 50% (20/40; P = 0.38). Oral fluid anti-VLP-16 IgG prevalence in HPV-16 DNA positive women was 28.6% (8/28) compared with 40% (16/40) in oral fluid from HPV-16 DNA negative women (P = 0.3). Amongst HPV-16 DNA positive women, the anti-VLP-16 IgG seroprevalence was 75% (21/28) and IgA seroprevalence 35.7% (10/28) and for the HPV-16 DNA negative women these values were 60% (24/40) and 32.5% (13/40), respectively. Oral IgA antibody testing proved no more sensitive than serum antibody detection for the determination of HPV infection but could be useful as a non-invasive screening method for women with cervical neoplasia and for estimating the mucosal antibody response to HPV vaccines.

Human papillomavirus virus-like particles are efficient oral immunogens when coadministered with Escherichia coli heat-labile enterotoxin mutant R192G or CpG DNA.

Certain human papillomaviruses (HPVs) cause most cervical cancer, which remains a significant source of morbidity and mortality among women worldwide. HPV recombinant virus-like particles (VLPs) are promising vaccine candidates for controlling anogenital HPV disease and are now being evaluated as a parenteral vaccine modality in human subjects. Vaccines formulated for injection generally are more costly, more difficult to administer, and less acceptable to recipients than are mucosally administered vaccines. Since oral delivery represents an attractive alternative to parenteral injection for large-scale human vaccination, the oral immunogenicity of HPV type 11 (HPV-11) VLPs in mice was previously investigated; it was found that a modest systemic neutralizing antibody response was induced (R. C. Rose, C. Lane, S. Wilson, J. A. Suzich, E. Rybicki, and A. L. Williamson, Vaccine 17:2129-2135, 1999). Here we examine whether VLPs of other genotypes may also be immunogenic when administered orally and whether mucosal adjuvants can be used to enhance VLP oral immunogenicity. We show that HPV-16 and HPV-18 VLPs are immunogenic when administered orally and that oral coadministration of these antigens with Escherichia coli heat-labile enterotoxin (LT) mutant R192G (LT R192G) or CpG DNA can significantly improve anti-VLP humoral responses in peripheral blood and in genital mucosal secretions. Our results also suggest that LT R192G may be superior to CpG DNA in this ability. These findings support the concept of oral immunization against anogenital HPV disease and suggest that clinical studies involving this approach may be warranted.

The impact of human immunodeficiency virus type 1 status on human papillomavirus (HPV) prevalence and HPV antibodies in serum and cervical secretions.

Human immunodeficiency virus (HIV) type 1-infected (HIV-positive) and -uninfected (HIV-negative) sex workers were examined for the presence of cervical human papillomavirus (HPV) DNA. Cervicovaginal rinse and serum samples from these women were examined for IgG and IgA antibodies to HPV-16 virus-like particles (VLP-16) by ELISA. The HIV-positive women displayed a significantly higher prevalence of HPV DNA (40/47 [85%]) than did the HIV-negative women (22/52 [42%]; P=.00001). Both HIV-positive and HIV-negative sex workers displayed a high seroprevalence rate for anti-VLP-16 IgG antibodies (27/40 [68%] and 30/43 [70%], respectively), but significantly fewer HIV-positive women than HIV-negative women had anti-VLP-16 serum IgA (6/40 [15%] vs. 17/43 [40%], respectively; P=.012). Significantly more HIV-positive women than HIV-negative women had cervical anti-VLP-16 IgG antibodies (16/49 [33%] vs. 6/63 [10%], respectively; P=.002) but not IgA antibodies (P=.3).

Immune and pathophysiological responses to different strains of Giardia duodenalis in neonatal mice.

Numerous studies have demonstrated various strain differences between Giardia isolates, but little is known about the immunology and pathogenesis of infections. This study aimed to compare host responses to strains of Giardi duodenalis differing in levels of virulence and pathogenicity and, by doing so, elucidate the mechanisms via which pathogenic strains establish infections. Marked differences were found in the infection dynamics, histopathological responses and serum antibody responses of neonatal mice infected with either G. duodenalis strain BRIS/83/HEPU/106 (isolated from a human) or BRIS/95/HEPU/2041 (isolated from a sulphur-crested cockatoo, Cacatua galerita). Infections with the bird strain were more intense (6.7-times greater) and persisted longer (by 14days) than infections with the human strain. The bird strain was more pathogenic and caused greater pathophysiological alteration to the gut mucosa, including increased villous atrophy, hyperplasia of goblet cells and vacuolated epithelial cells. Mice infected with the bird strain produced less serum anti-Giardia IgA and IgM, but more total (non-specific) serum IgA than those infected with the human strain of Giardia. This suggests that avian G. duodenalis strains are infective for mammalian hosts and may contribute to zoonotic infections. Furthermore, infection of mice with BRIS/95/HEPU/2041 serves as a good experimental model to provide further insight into the mechanisms via which G. duodenalis causes disease.