Pseudovirion-based Neutralization Assay Research Articles

Epidemiologic report and serologic findings for household contacts of three cases of influenza A (H7N9) virus infection

Background and objectiveWe conducted epidemiologic investigations and serologic assays on household contacts that were extensively exposed to three influenza A (H7N9) virus infected case-patients before infection-control practices were implemented. Study designData on the early clinical course of each patient and the exposure history for each patient's household contacts were obtained by interviewing household members and by reviewing medical records. Viral RNA in patient samples was tested using real-time reverse transcriptase polymerase chain reaction assay. Antibodies against H7N9 virus in serum samples were tested using hemagglutination inhibition and pseudovirus based neutralization assays. ResultsAll household contacts were extensively exposed to the case-patients without the use of measures to protect against infection. Viral RNA was detected in the specimens from case-patients for approximately 7–11 days after confirmation of infection. However, the results of the analyses of serum specimens taken from the household contacts 15–26 days post exposure revealed no evidence of transmission of H7N9 virus from the case-patients to the contacts. ConclusionDespite ample unprotected exposures to case-patients during the virus shedding period, household members in this report were not infected by the H7N9 virus.

Comparison of the Immunogenicity and Reactogenicity of Cervarix and Gardasil Human Papillomavirus Vaccines in HIV-Infected Adults: A Randomized, Double-Blind Clinical Trial

We compared the immunogenicity and reactogenicity of Cervarix or Gardasil human papillomavirus (HPV) vaccines in adults infected with the human immunodeficiency virus (HIV). This was a double-blind, controlled trial randomizing HIV-positive adults to receive 3 doses of Cervarix or Gardasil at 0, 1.5, and 6 months. Immunogenicity was evaluated for up to 12 months. Neutralizing anti-HPV-16/18 antibodies were measured by pseudovirion-based neutralization assay. Laboratory tests and diary cards were used for safety assessment. The HPV-DNA status of the participants was determined before and after immunization. Ninety-two participants were included in the study. Anti-HPV-18 antibody titers were higher in the Cervarix group compared with the Gardasil group at 7 and 12 months. No significant differences in anti-HPV-16 antibody titers were found among vaccine groups. Among Cervarix vaccinees, women had higher anti-HPV-16/18 antibody titers compared to men. No sex-specific differences in antibody titers were found in the Gardasil group. Mild injection site reactions were more common in the Cervarix group than in the Gardasil group (91.1% vs 69.6%; P = .02). No serious adverse events occurred. Both vaccines were immunogenic and well tolerated. Compared with Gardasil, Cervarix induced superior vaccine responses among HIV-infected women, whereas in HIV-infected men the difference in immunogenicity was less pronounced.

High-Throughput Pseudovirion-Based Neutralization Assay for Analysis of Natural and Vaccine-Induced Antibodies against Human Papillomaviruses

A highly sensitive, automated, purely add-on, high-throughput pseudovirion-based neutralization assay (HT-PBNA) with excellent repeatability and run-to-run reproducibility was developed for human papillomavirus types (HPV) 16, 18, 31, 45, 52, 58 and bovine papillomavirus type 1. Preparation of 384 well assay plates with serially diluted sera and the actual cell-based assay are separated in time, therefore batches of up to one hundred assay plates can be processed sequentially. A mean coefficient of variation (CV) of 13% was obtained for anti-HPV 16 and HPV 18 titers for a standard serum tested in a total of 58 repeats on individual plates in seven independent runs. Natural antibody response was analyzed in 35 sera from patients with HPV 16 DNA positive cervical intraepithelial neoplasia grade 2+ lesions. The new HT-PBNA is based on Gaussia luciferase with increased sensitivity compared to the previously described manual PBNA (manPBNA) based on secreted alkaline phosphatase as reporter. Titers obtained with HT-PBNA were generally higher than titers obtained with the manPBNA. A good linear correlation (R2 = 0.7) was found between HT-PBNA titers and anti-HPV 16 L1 antibody-levels determined by a Luminex bead-based GST-capture assay for these 35 sera and a Kappa-value of 0.72, with only 3 discordant sera in the low titer range. In addition to natural low titer antibody responses the high sensitivity of the HT-PBNA also allows detection of cross-neutralizing antibodies induced by commercial HPV L1-vaccines and experimental L2-vaccines. When analyzing the WHO international standards for HPV 16 and 18 we determined an analytical sensitivity of 0.864 and 1.105 mIU, respectively.

Kinetic and HPV infection effects on cross-type neutralizing antibody and avidity responses induced by Cervarix®

BackgroundWe previously demonstrated that Cervarix® elicits antibody responses against vaccine-related types for which clinical efficacy was demonstrated (HPV-31 and -45). Here, we evaluated the kinetics of neutralization titers and avidity of Cervarix®-induced antibodies up to 36 months of follow-up in unexposed and HPV infected women. MethodsA subset of women who participated in the Cost Rica HPV-16/18 Vaccine Trial had pre- and post-vaccination sera tested for antibody responses to HPV-16, -18, -31, -45, and -58 using a pseudovirion-based neutralization assay, and HPV-16 antibody avidity using an HPV-16 L1 VLP (virus-like particle)-based ELISA developed in our laboratory. ResultsIn uninfected women, neutralizing antibody titers did not reach significance until after the 3rd dose for HPV-31 (month 12, p=0.009) and HPV-45 (month 12, p=0.003), but then persisted up to month 36 (HPV-31, p=0.01; HPV-45, p=0.002). Individuals infected with HPV-16 or HPV-31 at enrollment developed a significantly higher median antibody response to the corresponding HPV type after one dose, but there was not a difference between median titers after three doses compared to the HPV negative group. Median HPV-16 antibody avidity and titer increased over time up to month 12; however, the HPV-16 avidity did not correlate well with HPV-16 neutralizing antibody titers at each time point examined, except for month 6. The median avidity levels were higher in HPV-16 infected women at month 1 (p=0.04) and lower in HPV-16 infected women at month 12 (p=0.006) compared to the HPV negative women. ConclusionsThe persistence of cross-neutralization titers at month 36 suggests cross-reactive antibody responses are likely to persist long-term and are not influenced by infection status at enrollment. However, the weak correlation between avidity and neutralization titers emphasizes the need for examining avidity in efficacy studies to determine if high avidity antibodies play a critical role in protection against infection.

Human Papillomavirus (HPV) L1 and L1-L2 Virus-Like Particle-Based Multiplex Assays for Measurement of HPV Virion Antibodies

Humoral immunity to human papillomavirus (HPV) has not been fully characterized, and there is currently no standard serologic test for the measurement of HPV antibodies. Most HPV serologic assays developed to date are based on virus-like particles (VLPs) of the major HPV capsid protein, L1. We sought to compare the performance of a multiplex HPV L1 VLP-based serologic assay to that of an assay based on VLPs comprised of both L1 and the minor capsid, L2. We developed HPV L1 VLP and L1-L2 VLP-based multiplex seroassays for the detection of HPV type 16 (HPV16) and HPV18 virion binding antibodies using Luminex fluorescent bead technology. We compared the performance of these assays to that of established pseudovirion-based neutralization and L1 VLP-based enzyme-linked immunosorbent assays (ELISAs). A total of 391 serum specimens from unvaccinated adult males and females were tested. The L1 and L1-L2 VLP multiplex seroassays each demonstrated substantial agreement with both the neutralization assays and the ELISAs for the detection of HPV16 antibodies (κ = 0.60 to 0.64). However, the L1-L2 VLP seroassay demonstrated better agreement with neutralization assays for the detection of HPV18 antibodies than the L1 VLP seroassay (κ = 0.74 and 0.43, respectively). L1 and L1-L2 VLP seroassays showed excellent agreement with one another for the detection of HPV16 antibodies (κ = 0.86) but only moderate agreement for HPV18 antibodies (κ = 0.44). The HPV L1-L2 VLP seroassay performs well for the concurrent measurement of HPV16 and -18 antibodies in large numbers of samples and may be extended to include other HPV types.

Comparison of the immunogenicity of the human papillomavirus (HPV)-16/18 vaccine and the HPV-6/11/16/18 vaccine for oncogenic non-vaccine types HPV-31 and HPV-45 in healthy women aged 18–45 years

Protection against oncogenic non-vaccine types (cross-protection) offered by human papillomavirus (HPV) vaccines may provide a significant medical benefit. Available clinical efficacy data suggest the two licensed vaccines (HPV-16/18 vaccine, GlaxoSmithKline Biologicals [GSK], and HPV-6/11/16/18 vaccine, Merck & Co., Inc.) differ in terms of protection against oncogenic non-vaccine HPV types -31/45. The immune responses induced by the two vaccines against these two non-vaccine HPV types (cross-reactivity) was compared in an observer-blind study up to Month 24 (18 months post-vaccination), in women HPV DNA-negative and seronegative prior to vaccination for the HPV type analyzed (HPV-010 [NCT00423046]). Geometric mean antibody titers (GMTs) measured by pseudovirion-based neutralization assay (PBNA) and enzyme-linked immunosorbent assay (ELISA) were similar between vaccines for HPV-31/45. Seropositivity rates for HPV-31 were also similar between vaccines; however, there was a trend for higher seropositivity with the HPV-16/18 vaccine (13.0–16.7%) versus the HPV-6/11/16/18 vaccine (0.0–5.0%) for HPV-45 with PBNA, but not ELISA. HPV-31/45 cross-reactive memory B-cell responses were comparable between vaccines. Circulating antigen-specific CD4+ T-cell frequencies were higher for the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (HPV-31 [geometric mean ratio [GMR] =2.0; p=0.0002] and HPV-45 [GMR=2.6; p=0.0092]), as were the proportion of T-cell responders (HPV-31, p=0.0009; HPV-45, p=0.0793). In conclusion, immune response to oncogenic non-vaccine HPV types -31/45 was generally similar for both vaccines with the exception of T-cell response which was higher with the HPV-16/18 vaccine. Considering the differences in cross-protective efficacy between the two vaccines, the results might provide insights into the underlying mechanism(s) of protection.

Comparative immunogenicity and safety of human papillomavirus (HPV)-16/18 vaccine and HPV-6/11/16/18 vaccine

In this observer-blind study (NCT00423046), women (N=1,106), stratified by age (18–26, 27–35, 36–45 years), were randomized (1:1) to receive the HPV-16/18 vaccine (Cervarix®, GlaxoSmithKline Biologicals, Months 0,1,6) or the HPV-6/11/16/18 vaccine (Gardasil® Merck & Co., Inc., Months 0,2,6). Month 7 results were previously reported; we now report Month 24 results. In the according-to-protocol cohort for immunogenicity (seronegative and DNA-negative at baseline for HPV type analyzed), seropositivity rates of neutralizing antibodies (nAbs) [pseudovirion-based neutralization assay] were, across all age strata, 100% (HPV-16/18 vaccine) and 97.5–100% (HPV-6/11/16/18 vaccine) for HPV-16, and 99.0–100% (HPV-16/18 vaccine) and 72.3–84.4% (HPV-6/11/16/18 vaccine) for HPV-18. Corresponding geometric mean titers (GMTs) were 2.4–5.8-fold higher for HPV-16 and 7.7–9.4-fold higher for HPV-18 with the HPV-16/18 vaccine versus the HPV-6/11/16/18 vaccine; HPV-16 and HPV-18 GMTs were significantly higher with the HPV-16/18 vaccine than the HPV-6/11/16/18 vaccine (p<0.0001) in the total vaccinated cohort (received ≥1 vaccine dose, irrespective of baseline sero/DNA-status). Similar results were obtained using enzyme-linked immunosorbent assay (ELISA). Positivity rates and GMTs of antigen-specific IgG antibodies in cervicovaginal secretions (ELISA) were not significantly different between vaccines. At Month 24, CD4+ T-cell responses for HPV-16 and HPV-18 were higher with the HPV-16/18 vaccine; memory B-cell response was higher for HPV-18 with the HPV-16/18 vaccine and similar between vaccines for HPV-16. Both vaccines were generally well tolerated. Although an immunological correlate of protection has not been defined, differences in the magnitude of immune response between vaccines may represent determinants of duration of protection.

Broad neutralizing antibody response and genetic variation in HIV-1 env genes in Koreans with primary HIV-1 infections

To determine the neutralization profiles induced by HIV-1 Korean clade B, which has a monophyletic lineage and relative limited genetic diversity, we investigated the ability of HIV variants to elicit neutralizing antibodies in the immune response to primary infection. We selected seven Korean drug-naïve subjects with an HIV-1 primary infection and did pseudovirion-based neutralization assays using env genes of Korean HIV origin. The neutralizing antibody responses to the Korean clade B showed broad reactivity to subtype B but a highly subtype-specific pattern. The lengths of the amino acid sequences and the PNGS numbers in the V1-V5 region were positively correlated with neutralization. These results imply that the genetic characteristics of HIV-1 env may affect neutralizing antibody responses in HIV-1-infected individuals. This is the first report describing the relationship between neutralizing antibody responses and HIV-1 genetic characteristics in Korean subjects. It can be useful for developing AIDS vaccines against HIV-1 subtype B strains.

Identification of a Dendrimeric Heparan Sulfate-Binding Peptide That Inhibits Infectivity of Genital Types of Human Papillomaviruses

Peptide dendrimers consist of a peptidyl branching core and/or covalently attached surface functional units. They show a variety of biological properties, including antiviral activity. In this study, a minilibrary of linear, dimeric, and dendrimeric peptides containing clusters of basic amino acids was evaluated for in vitro activity against human papillomaviruses (HPVs). The peptide dendrimer SB105-A10 was found to be a potent inhibitor of genital HPV types (i.e., types 16, 18, and 6) in pseudovirus-based neutralization assays. The 50% inhibitory concentration was between 2.8 and 4.2 μg/ml (0.59 and 0.88 μM), and no evidence of cytotoxicity was observed. SB105-A10 interacts with immobilized heparin and with heparan sulfates exposed on the cell surface, most likely preventing virus attachment. The findings from this study indicate SB105-A10 to be a leading candidate compound for further development as an active ingredient of a topical microbicide against HPV and other sexually transmitted viral infections.

Detection of HPV types and neutralizing antibodies in women with genital warts in Tianjin City, China

The serum samples and corresponding cervical swabs were collected from 50 women with genital warts from Tianjin city, China. The neutralizing antibodies against HPV-16, -18, -58, -45, -6 and -11 in serum samples were tested by using pseudovirus-based neutralization assays and HPV DNAs in cervical swabs were also tested by using a typing kit that can detect 21 types of HPV. The results revealed that 36% (18/50) of sera were positive for type-specific neutralizing antibodies with a titer range of 160-2560, of which 22%(11/50), 12%(6/50), 10%(5/50), 4%(2/50), 4%(2/50) and 2%(1/50) were against HPVs -6, -16, -18, -58, -45 and -11, respectively. Additionally, 60% (30/50) of samples were HPV DNA-positive, in which the most common types detected were HPV-68(18%), HPV-16(14%), HPV-58(12%), HPV-33(8%) and HPV-6, HPV-11, HPV-18 and HPV-52 (6% each). The concordance between HPV DNA and corresponding neutralizing antibodies was 56% (28/50) with a significant difference (P<0.05). The full-length sequences of five HPV types (HPV -42, -52, -53, -58 and -68) were determined and exhibited 98%-100% identities with their reported genomes. The present data may have utility for investigating the natural history of HPV infection and promote the development of HPV vaccines.

Comparison of the immunogenicity and safety of Cervarix™ and Gardasil® human papillomavirus (HPV) cervical cancer vaccines in healthy women aged 18–45 years

This observer-blind study compared the prophylactic human papillomavirus (HPV) vaccines, Cervarix™ (GlaxoSmithKline) and Gardasil® (Merck), by assessing immunogenicity and safety through one month after completion of the three-dose vaccination course. Women (n=1106) were stratified by age (18–26, 27–35, 36–45 years) and randomized (1:1) to receive Cervarix™ (Months 0, 1, 6) or Gardasil® (Months 0, 2, 6). At Month 7 after first vaccination, all women in the according-to-protocol cohort who were seronegative/DNA negative before vaccination for the HPV type analyzed had seroconverted for HPV-16 and HPV-18 serum neutralizing antibodies, as measured by pseudovirion-based neutralization assay (PBNA), except for two women aged 27–35 years in the Gardasil® group who did not seroconvert for HPV-18 (98%). Geometric mean titers of serum neutralizing antibodies ranged from 2.3–4.8-fold higher for HPV-16 and 6.8–9.1-fold higher for HPV-18 after vaccination with Cervarix™ compared with Gardasil®, across all age strata. In the total vaccinated cohort (all women who received at least one vaccine dose, regardless of their serological and DNA status prior to vaccination), Cervarix™ induced significantly higher serum neutralizing antibody titers in all age strata (p

Detection of HPV types and neutralizing antibodies in Gansu province, China

A total of 82 samples from patients with cervical cancer (Group 1) and 50 samples from patients with other genital diseases (Group 2) were collected in Gansu, China. All 132 samples were tested for HPV DNA with a typing kit that can detect 21 types of HPV, and also tested for neutralizing antibodies against HPV-16, -18, -58, -45, -6, and -11 using pseudovirus-based neutralization assays. The results revealed that 28% (23/82) of sera in Group 1 were positive for type-specific neutralizing antibodies with a titer range of 160-640, of which 23.2% (19/82), 2.4% (2/82), 2.4% (2/82), 1.2% (1/82), and 1.2% (1/82) were against HPV-16, -58, -6, -18, and -45, respectively. Only one serum (2%) in Group 2 was positive for neutralizing antibodies, which were against HPV-6 with a titer of 2,560. Overall, 85.4% (70/82) of samples in Group 1 were HPV DNA-positive, compared with 28% (14/50) of samples in Group 2. The seven most common types detected in Group 1 were HPV-16 (80%), HPV-52 (7.1%), HPV-66 and HPV-11 (5.7% each), and HPV-58, HPV-18, and HPV-33 (4.3% each), while the four most common types in Group 2 were HPV-16 (12%), HPV-52 and HPV-11 (6% each), and HPV-68 (4%). The concordance between HPV DNA and corresponding neutralizing antibodies was 32.9% (27/82) with a significant difference (P < 0.005). More specifically, the concordance was 42.7% (35/82) for HPV-16 in Group 1. The full-length sequences of six HPV types (HPV-16, -58, -33, -59, -11, and -68) were determined and showed 99% identities with their reported genomes.

Correlation between direct ELISA, single epitope-based inhibition ELISA and Pseudovirion-based neutralization assay for measuring anti-HPV-16 and anti-HPV-18 antibody response after vaccination with the AS04-adjuvanted HPV-16/18 cervical cancer vaccine

To monitor immune status during clinical trials and after vaccine registration, several assays have been developed to measure type-specific human papillomavirus (HPV) serum antibody levels. These include neutralization assays, single epitope-based inhibition immunoassays, and direct enzyme-linked immunosorbent assays (ELISAs). Neutralization assays based on multiple epitopes and independent of vaccine material are considered the ‘gold standard’ for unbiased assessment of the protective potential of vaccine-induced antibodies. However, their use in large clinical trials is challenging. Here, we compare both the direct ELISA and the single epitope-based inhibition ELISA with the pseudovirion-based neutralization assay (PBNA) for HPV-16/18 antibody responses in vaccinated women enrolled in trials of Cervarix™, GSK's cervical cancer vaccine. The direct ELISA, which is based on multiple epitopes, was shown to have a higher degree of sensitivity and correlation with the PBNA when compared with the single epitope-based inhibition ELISA. Among double-positive results, high correlations were observed between the PBNA and the direct ELISA (0.70-0.88 for HPV-16 and 0.82-0.94 for HPV-18) and also with the single epitope-based inhibition ELISA (0.60-0.89 for HPV-16 and 0.57-0.96 for HPV-18) in women aged 15-25 years. The correlation persisted up to 6.4 years after primary vaccination. Similar levels of correlation were observed for adolescents aged 10-14 years and women aged 46-55 years. Therefore, the direct ELISA appears to be an excellent surrogate for neutralizing activity and can be used to evaluate antibody response induced by L1 VLP-based cervical cancer vaccines, regardless of time elapsed after vaccination (up to 6.4 years) and the age of the vaccine recipient.

Genetic and Neutralization Properties of HIV-1 env Clones From Subtype B/BC/AE Infections in China

As HIV vaccines move into preclinical and clinical trials in China, pseudovirion-based neutralization assays, especially those using env genes of Chinese origin, are widely required to evaluate the ability of HIV vaccines to induce neutralizing antibody (nAb) responses. Functional gp160 genes from plasma samples from Chinese HIV-infected patients were cloned and sequenced and then used to establish a pseudovirus-based neutralization assay. The neutralization phenotypes of the Env-pseudotyped viruses were characterized with known nAbs (4E10, 2F5, IgG1b12, and 2G12) and 43 plasma samples from patients infected with different HIV subtypes. Overall, 27 functional gp160 genes (18 subtype BC, 3 subtype AE, and 6 subtype B) of HIV-1 were obtained, and their full-length nucleotide sequences were analyzed. The results confirmed the presence of significant genetic diversity among the clones. 4E10 neutralized all 27 Env-pseudotyped viruses, whereas IgG1b12 neutralized 44% of them. 2F5 neutralized 67% and 100% of subtype B and AE clones, respectively, but not subtype BC clones, whereas 2G12 neutralized 33% of subtype B viruses but not subtype BC and AE viruses. There were significant differences in the cross-neutralization activities when the neutralization phenotypes of the 27 Env-pseudotyped viruses were characterized using 43 HIV-positive plasma samples. These characterized functional HIV-1 env clones should be useful for standardizing neutralization assays in China.

Characterization of antibody response in women with naturally acquired HPV 16 infection using pseudovirion‐based neutralization assay

Antibody response to HPV16 infection has mainly been studied using Virus‐like Particle (VLP)‐based ELISA assays of serum. Local antibody response at the cervix may be a better indicator of protection. In this study, the pseudovirion–based neutralization assay was adapted to measure antibody titers in response to naturally acquired HPV 16 infection in both serum and cervical mucous samples. Samples were collected from women (n=230) attending colposcopy clinics and matched for age, race and HPV16 DNA status. Results showed that 54.1% of sera and 17.2% of mucous samples had positive antibody titers. Correlation of titers between the matched sera and mucosal samples was poor (ρ= 0.54), with sera being more likely to be positive for an antibody response (κ= 0.25). Univariate analysis of the data showed HPV 16 DNA status was significantly associated with both serum and mucosal antibody titer, but not with HPV 16 viral load or cervical disease. Comparison of the neutralization assay to the standard HPV 16 VLP‐based IgG ELISA also showed a fair correlation (ρ= 0.74) between titers and absorbance. In naturally acquired infections, serum antibodies are a more sensitive measure of exposure than mucosal antibodies. This study was supported in part by the National Cancer Institute's Early Detection Research Network Interagency Agreement Y1‐CN‐0101‐01.