Sara L. Bissett

A Randomized, Observer-Blinded Immunogenicity Trial of Cervarix® and Gardasil® Human Papillomavirus Vaccines in 12-15 Year Old Girls

Background The current generation of Human Papillomavirus (HPV) vaccines, Cervarix® and Gardasil®, exhibit a high degree of efficacy in clinical trials against the two high-risk (HR) genotypes represented in the vaccines (HPV16 and HPV18). High levels of neutralizing antibodies are elicited against the vaccine types, consistent with preclinical data showing that neutralizing antibodies can mediate type-specific protection in the absence of other immune effectors. The vaccines also confer protection against some closely related non-vaccine HR HPV types, although the vaccines appear to differ in their degree of cross-protection. The mechanism of vaccine-induced cross-protection is unknown. This study sought to compare the breadth and magnitudes of neutralizing antibodies against non-vaccine types elicited by both vaccines and establish whether such antibodies could be detected in the genital secretions of vaccinated individuals. Methods and Findings Serum and genital samples were collected from 12–15 year old girls following vaccination with either Cervarix® (n = 96) or Gardasil® (n = 102) HPV vaccine. Serum-neutralizing antibody responses against non-vaccine HPV types were broader and of higher magnitude in the Cervarix®, compared to the Gardasil®, vaccinated individuals. Levels of neutralizing and binding antibodies in genital secretions were closely associated with those found in the serum (r = 0.869), with Cervarix® having a median 2.5 (inter-quartile range, 1.7–3.5) fold higher geometric mean HPV-specific IgG ratio in serum and genital samples than Gardasil® (p = 0.0047). There was a strong positive association between cross-neutralizing antibody seropositivity and available HPV vaccine trial efficacy data against non-vaccine types. Conclusions These data demonstrate for the first time that cross-neutralizing antibodies can be detected at the genital site of infection and support the possibility that cross-neutralizing antibodies play a role in the cross-protection against HPV infection and disease that has been reported for the current HPV vaccines. Trial Registration ClinicalTrials.gov NCT00956553

Development and optimization of an internally controlled dried blood spot assay for surveillance of human immunodeficiency virus type-1 drug resistance

OBJECTIVES We present the evaluation of a methodology for the genotypic assessment of human immunodeficiency virus type-1 (HIV-1) drug resistance, optimized for use with dried blood spots (DBS). METHODS The ability to generate HIV-1 protease (PR) and reverse transcriptase (RT) contiguous amplicons and nucleotide sequences from DBS was evaluated. Different collection matrices and extraction methodologies were compared. The relative subtype sensitivity of the amplification strategy was assessed using a comprehensive panel of plasmids representing A-H subtypes. A panel of DBS and plasma specimens was subjected to HIV genotyping. Sequences generated from each sample type were compared. RESULTS Extensive replicate testing revealed most sensitivity with the use of 903 filter paper and silica/guanidine extraction, which had an estimated 95% inclusivity endpoint of 1542 proviral copies/mL, as compared with 21 573 proviral copies/mL for the FTA system. All HIV-1 group M subtypes analysed-with the exception of subtypes A2, AE, AG, F and H-had a relative sensitivity of </=10 plasmid copies/PCR reaction. The PCR was multiplexed to include amplification of a human housekeeping gene to monitor the integrity of the human genomic DNA. Using a panel of clinical samples, we demonstrated the ability to amplify and sequence from 83% (n = 10) in the PR region and 100% (n = 12) in the RT region, of samples with detectable viral load. All specimens with an HIV-1 RNA load >/=1000 copies/mL were successfully amplified and sequenced. Twelve specimens had pol genotyping from both plasma and DBS samples. Sequence analysis and drug resistance interpretation revealed that 10 (83%) provided concordant drug resistance interpretation. CONCLUSIONS Our results demonstrate that the technique is appropriate for surveillance of drug resistance in untreated individuals and those with virological failure on therapy.

Neutralization of non-vaccine human papillomavirus pseudoviruses from the A7 and A9 species groups by bivalent HPV vaccine sera

Highlights ► We examined neutralizing antibody in girls immunized with bivalent HPV vaccine. ► Antibody cross-reactivity dependent on response to vaccine types. ► Significant cross-reactivity against HPV31, HPV33, HPV45. ► Cross-reactive levels are low at <1% of vaccine type antibody responses. ► Potential role for cross-reactive antibodies as surrogate of protection.

Human papillomavirus genotype detection and viral load in paired genital and urine samples from both females and males.

The ability to detect type‐specific high risk HPV (HR‐HPV) infections in samples from females and males is important for monitoring the epidemiology of HPV and the impact of vaccination. Type‐specific detection concordance between paired urine and genital samples from females (n = 264) undergoing routine colposcopy and males (n = 88) attending a genito‐urinary medicine clinic was evaluated using an in‐house genotyping assay. The overall inter‐rater agreement (κ) was 0.781 for female pairs and 0.346 for male pairs. Female urine had sensitivity for detection of HPV16/18 and HR‐HPV of 75% and 84%, respectively, while male urine had sensitivities of 13% and 28%, respectively. Genital samples had a higher HPV DNA copy number than urine although a small proportion (10%) of urine samples had a higher copy number than the corresponding genital sample. The proportion of females with normal cytology positive for HPV16/18 was 19%, increasing to 57% in moderate or severely dyskaryotic samples. The same trend was seen in the corresponding urine (19–43%) compounded by the reduced sensitivity of this sample type. The HPV16 viral load in female genital samples, but not in urine, was weakly associated with cervical disease stage. Despite reduced sensitivity, urine appears to be an appropriate surrogate sample for type‐specific HPV detection in females for epidemiological objectives. The lower sensitivity and lack of association between viral load and disease stage in urine suggest that urine may not be useful for clinical management of HPV infection. The utility of urine for type‐specific detection in males is less certain. J. Med. Virol. 83:1744–1751, 2011.

Systemic and mucosal immune responses to sublingual or intramuscular human papilloma virus antigens in healthy female volunteers.

The sublingual route has been proposed as a needle-free option to induce systemic and mucosal immune protection against viral infections. In a translational study of systemic and mucosal humoral immune responses to sublingual or systemically administered viral antigens, eighteen healthy female volunteers aged 19–31 years received three immunizations with a quadravalent Human Papilloma Virus vaccine at 0, 4 and 16 weeks as sublingual drops (SL, n = 12) or intramuscular injection (IM, n = 6). IM antigen delivery induced or boosted HPV-specific serum IgG and pseudovirus-neutralizing antibodies, HPV-specific cervical and vaginal IgG, and elicited circulating IgG and IgA antibody secreting cells. SL antigens induced ∼38-fold lower serum and ∼2-fold lower cervical/vaginal IgG than IM delivery, and induced or boosted serum virus neutralizing antibody in only 3/12 subjects. Neither route reproducibly induced HPV-specific mucosal IgA. Alternative delivery systems and adjuvants will be required to enhance and evaluate immune responses following sublingual immunization in humans. Trial Registration ClinicalTrials.gov NCT00949572

Cross-neutralizing antibodies elicited by the Cervarix® human papillomavirus vaccine display a range of Alpha-9 inter-type specificities.

Highlights • We explored Cervarix® HPV vaccine cross-reactive antibody specificity.• L1 VLP binding was a poor surrogate for L1L2 pseudovirus neutralization specificity.• Cross-neutralizing antibodies comprise a small proportion of total antibody.• Multiple, overlapping cross-neutralizing antibody specificities exist.

Amino acid sequence diversity of the major human papillomavirus capsid protein: Implications for current and next generation vaccines

Highlights • We evaluated amino acid diversity of the major capsid protein of HPV.• Residues displaying high entropy were found within surface-exposed domains.• We discuss the implications of this diversity on the current and next generation HPV vaccines.

The 'Red Queen' dilemma - running to stay in the same place: reflections on the evolutionary vector of HBV in humans

BACKGROUND Estimates for the evolutionary rate of HBV until now have been interpreted as showing that HBV is a relatively recent acquisition for mankind. The existence of defined HBV genotypes is thought to represent past founder effects. We have explored virus mutation in a group of 48 persistently infected blood donors sampled at two points in time and suggest otherwise. METHODS HBV-infected donors were detected by hepatitis B surface antigen (HBsAg) screening and staged by hepatitis B e markers. Serum DNA from those persistently infected with HBV was characterized by consensus sequencing and the amino acid sequences inferred. These were compared against consensus genotype sequences and divergence measured at two points in time. RESULTS Rates of viral mutation were higher across both HBsAg and hepatitis B core antigen in the group of donors seropositive for hepatitis B e antibody (1.36×10⁻³ and 1.54×10⁻³ changes per residue per year, respectively) than in those seropositive for hepatitis B e antigen (4.59×10⁻⁴ and 6.62×10⁻⁴ changes per residue per year, respectively). Codon mutations reverting to the genotype consensus were commonly seen. Codon changes were clustered close to the C-terminal region of HBsAg and were accommodated in overlapping polymerase by synonymous substitutions. CONCLUSIONS It is suggested that in vivo HBV behaves as a self-normalizing meme and mutational rates, although high, do not lead to significant change over time in a persistent infection. This would be compatible with co-evolution within its human host and introduction within humans being an ancient occurrence.

Human papillomavirus antibody reference reagents for use in postvaccination surveillance serology.

ABSTRACT Suitably controlled serosurveillance surveys are essential for evaluating human papillomavirus (HPV) immunization programs. A panel of plasma samples from 18-year-old females was assembled, the majority of the samples being from recipients of the bivalent HPV vaccine. Antibody specificities were evaluated by three independent laboratories, and 3 pools that displayed no antibodies to any HPV type tested or intermediate or high levels of antibody to HPV16, HPV18, HPV31, and HPV45 were created. These pools will be useful as control reagents for HPV serology.

Pre-clinical immunogenicity of human papillomavirus alpha-7 and alpha-9 major capsid proteins

Highlights • Comprehensive pre-clinical immunogenicity evaluation of HPV L1 major capsid protein.• Majority neutralizing antibody response was genotype-specific.• Reciprocal cross-neutralization between some Alpha-7 and Alpha-9 genotypes.• Tetravalent formulation (HPV16/18/39/58) induced broadly neutralizing antibodies.• These data improve our understanding of the antigenic diversity of the L1 protein.