Explore the vast range of titles available.

To re-optimize the pneumococcal (Pn) electrochemiluminescence (ECL) assay and to validate and bridge the enhanced assay to the WHO ELISA, to support the Phase III clinical trial program for V114, a 15-valent Pn conjugate vaccine. The Pn ECL assay was re-optimized, validated and formally bridged to the WHO ELISA. The enhanced Pn ECL assay met all prespecified validation acceptance criteria and demonstrated concordance with the WHO ELISA. The corresponding threshold value remains at 0.35 μg/ml for all 15 serotypes. The enhanced Pn ECL assay has been validated for the measurement of antibodies to 15 Pn capsular polysaccharides and is concordant with the WHO ELISA, supporting its use in clinical trials.

To streamline and improve throughput, the agar-based multiplexed opsonophagocytic killing assay (MOPA) was optimized and validated on a microcolony platform for use in the Phase III clinical trial program for V114, an MSD 15-valent pneumococcal conjugate vaccine candidate. The precision, dilutional linearity and specificity of the microcolony MOPA (mMOPA) were assessed for each serotype in validation experiments. All prespecified acceptance criteria on assay performance were satisfied. Accuracy was assessed by testing 007sp and the US FDA reference panel and comparing to consensus values. The mMOPA produced comparable results to other opsonophagocytic killing assays/MOPAs. The mMOPA is suitable for measuring functional antibodies in adult and pediatric samples. Benefits include throughput, reduced analyst-to-analyst variability and automation potential.

Background Pneumococcal conjugate vaccines (PCVs) are designed to induce serotype-specific antibodies; however, antibodies raised against pneumococcal polysaccharides in the vaccine can also cross-react to other serotypes based on structural similarities. Cross-reactivity has been demonstrated for some licensed PCVs and is a worthy consideration to broaden protection against pneumococcal disease. This secondary analysis examined cross-reactive immune responses within serogroups 6 and 15 following vaccination with V116, an adult-specific PCV. Methods Antibody levels were assessed across five phase 3 clinical studies at baseline and 30 days following vaccination. Results V116 elicits functional antibodies to vaccine serotypes 6A and 15C that result in cross-reactive antibodies to serotypes 6C and 15B, respectively, as assessed by opsonophagocytic activity responses from pre- to post-vaccination and IgG geometric mean concentrations. Conclusions V116-induced antibodies demonstrate cross-reactivity to serotypes 6C and 15B, suggesting protective immune responses may be raised against these non-vaccine serotypes. Plain language summary V116 is a vaccine designed for adults to prevent bacterial infections caused by pneumococcus, such as pneumonia and meningitis. Following vaccination with V116, the body produces antibodies that help neutralize pneumococcus. As part of the clinical evaluation of V116, this study examined how antibodies generated by the vaccine cross-react with certain types of pneumococcus not contained in the vaccine. Immune responses were generated against pneumococcal types not present in the vaccine, suggesting V116 may provide protection against these pneumococcal types as well. These findings support the value of vaccination with V116 to prevent pneumococcal disease. Li, Platt et al. examine cross-reactivity of antibodies induced by the adult-specific pneumococcal conjugate vaccine V116 within serogroups 6 and 15. Antibodies demonstrated cross-reactivity to non-vaccine serotypes 6 C and 15B, suggesting broader coverage may be available beyond the 21 V116 serotypes following vaccination.

Immunogenicity of pneumococcal vaccines is measured using post-vaccination serotype-specific immunoglobulin G (IgG) antibodies in serum using enzyme-linked immunoassays with the 007sp reference serum containing serotype-specific IgG for 24 pneumococcal serotypes. With the development of next-generation PCVs, a new S. pneumoniae reference serum standard was needed to include serotypes beyond the existing 24 in 007sp. In this study, antibody concentrations to 33 pneumococcal serotypes were assigned in a new Merck Pneumococcal Reference Serum Standard (MPRSS-01) using the pneumococcal electrochemiluminescence assay, enabling V116 to maintain the link to the historical human pneumococcal standard reference serum while utilizing the new human pneumococcal reference serum.

Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. Two multiplex immunoassays are routinely used to assess antibody responses in clinical trials of the 9-valent human papillomavirus (9vHPV) vaccine. The HPV6/11/16/18/31/33/45/52/58 competitive Luminex immunoassay (HPV-9 cLIA) and HPV6/11/16/18/31/33/45/52/58 total immunoglobulin G Luminex immunoassay are used for measurements of immunogenicity. Following their initial validation in 2010, both assays were redeveloped, and several parameters were optimized, including the coating concentration of virus-like particles, type of Luminex microspheres, serum sample and reference standard diluent, reference standard starting dilution and titration series, and vendor and concentration of the phycoerythrin-labeled antibodies. Validation studies evaluated the assay performance parameters, including the intra-assay precision (repeatability), intermediate precision, linearity, relative accuracy, and limits of quantitation. In addition, since maintaining a link to the original assays that were used in trials supporting vaccine licensure is critical, the assays were formally bridged to the previous assay versions by using individual patient sera from a 9vHPV vaccine clinical trial ( n  = 150 day 1 [prevaccination] samples; n  = 100 month 7 [1 month post-last vaccine dose] and n  = 100 month 36 [30 months post-last vaccine dose; antibody persistence] samples). The results of the validation studies indicate that both optimized assays are accurate, specific, and precise over their respective quantifiable ranges. There was a strong linear association between the new and previous versions of both assays. Assay serostatus cutoffs for the redeveloped assays were established based on the bridging studies and, for the HPV-9 cLIA, further refined, based on additional data from HPV vaccine clinical studies so as to align the seropositivity rates between assay versions. IMPORTANCE Assay modernization is a key aspect of vaccine life cycle management. Thus, new, reoptimized versions of two 9vHPV immunoassays have been developed and validated for use in ongoing and future HPV vaccine clinical trials. These assays are suitable for use in high-throughput testing for HPV antibodies in serum samples. Bridging to previous versions of the assays allows for the continuous monitoring of immune responses across assay versions, including in immunogenicity studies that involve new populations as well as long-term follow-up studies.

Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Streptococcus pneumoniae is a major cause of community-acquired pneumonia (CAP) in young children, older adults, and those with immunocompromised status. Since the introduction of pneumococcal vaccines, the burden of invasive pneumococcal disease caused by vaccine serotypes (STs) has decreased; however, the effect on the burden of CAP is unclear, potentially due to the lack of testing for pneumococcal STs. We describe the development, qualification, and clinical validation of a high-throughput and multiplex ST-specific urine antigen detection (SSUAD) assay to address the unmet need in CAP pneumococcal epidemiology. The SSUAD assay is sensitive and specific to the 15 STs in the licensed pneumococcal conjugate vaccine V114 (STs 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 22F, 23F, and 33F) and uses ST-specific monoclonal antibodies for rapid and simultaneous quantification of the 15 STs using a Luminex microfluidics system. The SSUAD assay was optimized and qualified using healthy adult urine spiked with pneumococcal polysaccharides and validated using culture-positive clinical urine samples ( n  = 34). Key parameters measured were accuracy, precision, sensitivity, specificity, selectivity, and parallelism. The SSUAD assay met all prespecified validation acceptance criteria and is suitable for assessments of disease burden associated with the 15 pneumococcal STs included in V114. IMPORTANCE Streptococcus pneumoniae has more than 90 serotypes capable of causing a range of disease manifestations, including otitis media, pneumonia, and invasive diseases, such as bacteremia or meningitis. Only a minority (<10%) of pneumococcal diseases are bacteremic with known serotype distribution. Culture and serotyping of respiratory specimens are neither routine nor reliable. Hence, the serotype-specific disease burden of the remaining (>90%) noninvasive conditions is largely unknown without reliable laboratory techniques. To address this need, a 15-plex urine antigen detection assay was developed and validated to quantify pneumococcal serotype-specific capsular polysaccharides in urine. This assay will support surveillance to estimate the pneumococcal disease burden and serotype distribution in nonbacteremic conditions. Data obtained from this assay will be critical for understanding the impact of pneumococcal vaccines on noninvasive pneumococcal diseases and to inform the choice of pneumococcal serotypes for next-generation vaccines.

The V1/V2 region and the V3 loop of the human immunodeficiency virus type I (HIV-1) envelope (Env) protein are targets for neutralizing antibodies and also play an important functional role, with the V3 loop largely determining whether a virus uses CCR5 (R5), CXCR4 (X4), or either coreceptor (R5X4) to infect cells. While the sequence of V3 is variable, its length is highly conserved. Structural studies indicate that V3 length may be important for interactions with the extracellular loops of the coreceptor. Consistent with this view, genetic truncation of the V3 loop is typically associated with loss of Env function. We removed approximately one-half of the V3 loop from three different HIV-1 strains, and found that only the Env protein from the R5X4 strain R3A retained some fusion activity. Loss of V1/V2 (DeltaV1/V2) was well tolerated by this virus. Passaging of virus with the truncated V3 loop resulted in the derivation of a virus strain that replicated with wild-type kinetics. This virus, termed TA1, retained the V3 loop truncation and acquired several adaptive changes in gp120 and gp41. TA1 could use CCR5 but not CXCR4 to infect cells, and was extremely sensitive to neutralization by HIV-1 positive human sera, and by antibodies to the CD4 binding site and to CD4-induced epitopes in the bridging sheet region of gp120. In addition, TA1 was completely resistant to CCR5 inhibitors, and was more dependent upon the N-terminal domain of CCR5, a region of the receptor that is thought to contact the bridging sheet of gp120 and the base of the V3 loop, and whose conformation may not be greatly affected by CCR5 inhibitors. These studies suggest that the V3 loop protects HIV from neutralization by antibodies prevalent in infected humans, that CCR5 inhibitors likely act by disrupting interactions between the V3 loop and the coreceptor, and that altered use of CCR5 by HIV-1 associated with increased sensitivity to changes in the N-terminal domain can be linked to high levels of resistance to these antiviral compounds.

To better inform pneumococcal immunization policies, ongoing surveillance for pneumococcal community-acquired pneumonia (CAP) is crucial. To estimate the serotype-specific CAP burden of pneumococcal disease following the introduction of a new 15-valent pneumococcal conjugate vaccine (PCV), V114, a 15-plex serotype-specific urine antigen detection (SSUAD) assay was developed as a tool for surveillance of Streptococcuspneumoniae serotypes. V114–017 (NCT03547167; EudraCT 2017–004915-38) was a phase 3 randomized controlled trial in which participants (18–49 years) received V114 or 13-valent PCV (PCV13; as an active comparator), followed 6 months later by 23-valent pneumococcal polysaccharide vaccine (PPSV23). Here, we report findings from a prespecified sub-study nested within the phase 3 trial that descriptively assessed the impact of nasopharyngeal/oropharyngeal (NP/OP) carriage and pneumococcal vaccination on serotype detection with the SSUAD assay. In total, 301 individuals (all American Indian/Alaska Native) participated in the sub-study. NP/OP and urine samples were collected at 10 timepoints between baseline (prior to vaccination) and Month 7 (30 days following vaccination with PPSV23). NP/OP carriage was determined using qualitative polymerase chain reaction for pneumococcus detection and serotyping, and urine samples were tested in parallel with SSUAD. At any timepoint, NP/OP carriage was <2.0 % for 10 of the V114 serotypes; carriage was ∼2.6 % for serotype 1 and ranged between 4.0 % and 7.0 % for serotypes 4, 5, 9V, and 33F. At baseline, serotype-specific pneumococcal polysaccharide antigens were detected by SSUAD in only six study participants for serotypes 19A, 19F, and 1. SSUAD positivity for serotypes 4, 5, and 9V increased transiently following vaccination with V114/PCV13 and PPSV23, while SSUAD positivity lasted the longest for serotype 19A following PPSV23 vaccination. In general, SSUAD positivity appeared unrelated to NP/OP carriage. Our findings suggest SSUAD can support pneumococcal disease surveillance and vaccine effectiveness research, excluding individuals with recent pneumococcal vaccination to avoid false-positives.

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-type Listeria monocytogenes ( Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuated Lm could serve as a vaccine vector. We sought to examine the potential of live attenuated Lm to induce cellular immune responses to HIV Gag. Rhesus macaques were immunized with Lmdd-gag that expresses HIV gag and lacks two genes in the d-alanine ( d-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenous d-ala. Lmdd-gag was given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting; d-ala was co-administered with all vaccinations. Lmdd-gag and d-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinant Lmdd-gag is safe and immunogenic in primates.

Induction of strong cellular immunity will be important for AIDS vaccine candidates. Natural infection with wild-typeListeria monocytogenes(Lm), an orally transmitted organism, is known to generate strong cellular immunity, thus raising the possibility that live attenuatedLmcould serve as a vaccine vector. We sought to examine the potential of live attenuatedLmto induce cellular immune responses to HIV Gag. Rhesus macaques were immunized withLmdd-gagthat expresses HIVgagand lacks two genes in thed-alanine (d-ala) synthesis pathway. Without this key component of the bacterial cell wall, vaccine vector replication critically depends on exogenousd-ala.Lmdd-gagwas given to animals either solely orally or by oral priming followed by intramuscular (i.m.) boosting;d-ala was co-administered with all vaccinations.Lmdd-gagandd-ala were well tolerated. Oral priming/oral boosting induced Gag-specific cellular immune responses, whereas oral priming/i.m. boosting induced systemic as well as mucosal anti-Gag antibodies. These results suggest that the route of vaccination may bias anti-Gag immune responses either towards T-helper type 1 (Th1) or Th2 responses; overall, our data show that live attenuated, recombinantLmdd-gagis safe and immunogenic in primates.