Heterologous immunization with Covishield and Pfizer vaccines against SARS-CoV-2 elicits a robust humoral immune response.

Understanding the efficacy and durability of heterologous immunization schedules against SARS-CoV-2 is critical, as supply demands and vaccine choices become significant issues in the global vaccination strategy. Here we characterize the neutralizing antibodies produced in two subjects who received combination immunizations against SARS-CoV-2, first with Covishield (Oxford-AstraZeneca) vaccine, followed 33 days later with a second dose (booster) shot of the Pfizer-BioNTech vaccine. Serum samples were collected 25 days following the primary vaccination and 13 days after the secondary Pfizer vaccination. Both subjects exhibited increased levels of isotype IgG and IgM antibodies directed against the entire spike protein following immunizations. These antibodies also exhibited increased reactivity with the receptor binding domain (RBD) in the spike protein and neutralized the infectivity of replicating vesicular stomatitis virus (VSV) that contains the COVID-19 coronavirus S protein gene in place of its normal G glycoprotein. This VSV pseudovirus also contains the reporter gene for enhanced green fluorescent protein (eGFP). Antibody titers against the spike protein and serum neutralization titers against the reporter virus are reported for the 2 heterologous vaccinated individuals and compared to a positive control derived from a convalescent patient and a negative control from an unexposed individual. The Pfizer-BioNTech vaccine increased antibody binding to the spike protein and RBD, and approached levels found in the convalescent positive control. Neutralizing antibodies against the VSV-S pseudovirus in the 2 subjects also approached levels in the convalescent sera. These results firmly validate the value of the Pfizer-BioNTech vaccine in boosting immunity following initial Covishield inoculation.

Multi-target plasmid controls for conventional and real-time PCR-based serotyping of Streptococcus pneumoniae.

BACKGROUND: Serotyping of Streptococcus pneumoniae is an integral part of disease surveillance, with over 92 serotypes characterized to date using traditional serotyping. To identify the most predominant disease causing serotypes, molecular serotyping methods are now increasingly being used, like conventional and real-time multiplex PCR (cmPCR and rmPCR, respectively). Given that cmPCR consists of eight reactions spanning 41 targets, and rmPCR consists of seven triplex reactions, standardizing positive controls for these assays is challenging. As such, a 43-target plasmid for cmPCR (pSpn-CM1) and a 23 target plasmid for rmPCR (pSpn-RM1) were designed and validated. METHODS: Plasmid pSpn-RM1 was designed and synthesized as chimeric DNA sequences to include all PCR target primer binding sites sequences for cmPCR. Plasmid pSpn-RM1 consisted of all primer and probe sequences required for rmPCR. Additional targets (lytA and cpsA) were included in both plasmids for quantification, following their propagation and purification from Escherichia coli. RESULTS: When tested using the cmPCR reactions, all targets could be reproducibly be detected using pSpn-CM1 as template, with good amplicon visibility at a concentration of 1.4 (± 0.3) × 105 copies/ml was used. For the rmPCR reactions, all targets were reproducibly amplified with a concentration of 1.1 (± 0.2) × 104 copies/ml of pSpn-RM1, and the PCR efficiency for each target was equivalent to DNA extracted from representative S. pneumoniae serotypes. CONCLUSIONS: These quantifiable multi-target plasmids simplify the preparation of controls for PCR-based serotyping of S. pneumoniae, and methods herein could be extended to other highly multiplexed PCR assays.

Adaptive Mutations in Influenza A/California/07/2009 Enhance Polymerase Activity and Infectious Virion Production.

Mice are not natural hosts for influenza A viruses (IAVs), but they are useful models for studying antiviral immune responses and pathogenesis. Serial passage of IAV in mice invariably causes the emergence of adaptive mutations and increased virulence. Here, we report the adaptation of IAV reference strain A/California/07/2009(H1N1) (also known as CA/07) in outbred Swiss Webster mice. Serial passage led to increased virulence and lung titers, and dissemination of the virus to brains. We adapted a deep-sequencing protocol to identify and enumerate adaptive mutations across all genome segments. Among mutations that emerged during mouse-adaptation, we focused on amino acid substitutions in polymerase subunits: polymerase basic-1 (PB1) T156A and F740L and polymerase acidic (PA) E349G. These mutations were evaluated singly and in combination in minigenome replicon assays, which revealed that PA E349G increased polymerase activity. By selectively engineering three PB1 and PA mutations into the parental CA/07 strain, we demonstrated that these mutations in polymerase subunits decreased the production of defective viral genome segments with internal deletions and dramatically increased the release of infectious virions from mouse cells. Together, these findings increase our understanding of the contribution of polymerase subunits to successful host adaptation.

PCR-based discrimination of emerging Streptococcus pneumoniae serotypes 22F and 33F.

Serotyping of Streptococcus pneumoniae is important to monitor disease epidemiology and assess the impact of pneumococcal vaccines. Traditionally, the Quellung reaction used serotype-specific antibodies to classify S. pneumoniae based on differences in capsular antigens. More recently, PCR-based serotype deduction relying on serotype-specific capsule biosynthesis genes has been broadly applied for pneumococcal surveillance. However, PCR-based serotyping lacks discrimination for certain S. pneumoniae serotypes, including the differentiation of serotype 22F from 22A, and serotype 33F from 33A and 37. Serotypes 22F and 33F are emerging serotypes that are absent in the currently licensed 13-valent pneumococcal conjugate vaccine, but present in the new candidate 15-valent formulation. This study validated novel PCR reactions to detect and discriminate S. pneumoniae serotypes 22F and 33F. In order to differentiate S. pneumoniae serotypes 22F or 33F from genetically similar serotypes, two novel PCR reactions were designed and validated. The specificity of all PCR targets was evaluated using all 92 different S. pneumoniae serotypes, as well as 32 other streptococci. Reproducibility was evaluated using geographically and genetically diverse strains of S. pneumoniae serotypes 22F and 22A, or serotypes 33F, 33A, and 37 that were previously characterized by reputable reference laboratories. Overall, S. pneumoniae serotypes 22F and 33F could be accurately and reproducibly be detected and discriminated using PCR alone. Such a molecular serotyping approach provides a valuable diagnostic tool that is feasible in any molecular laboratory, to enable pneumococcal serotype surveillance and subsequent assessment of the impact of the new 15-valent candidate pneumococcal vaccine.

Standardization of Hemagglutination Inhibition Assay for Influenza Serology Allows for High Reproducibility between Laboratories.

Standardization of the hemagglutination inhibition (HAI) assay for influenza serology is challenging. Poor reproducibility of HAI results from one laboratory to another is widely cited, limiting comparisons between candidate vaccines in different clinical trials and posing challenges for licensing authorities. In this study, we standardized HAI assay materials, methods, and interpretive criteria across five geographically dispersed laboratories of a multidisciplinary influenza research network and then evaluated intralaboratory and interlaboratory variations in HAI titers by repeatedly testing standardized panels of human serum samples. Duplicate precision and reproducibility from comparisons between assays within laboratories were 99.8% (99.2% to 100%) and 98.0% (93.3% to 100%), respectively. The results for 98.9% (95% to 100%) of the samples were within 2-fold of all-laboratory consensus titers, and the results for 94.3% (85% to 100%) of the samples were within 2-fold of our reference laboratory data. Low-titer samples showed the greatest variability in comparisons between assays and between sites. Classification of seroprotection (titer ≥ 40) was accurate in 93.6% or 89.5% of cases in comparison to the consensus or reference laboratory classification, respectively. This study showed that with carefully chosen standardization processes, high reproducibility of HAI results between laboratories is indeed achievable.

Does sporadic Listeria gastroenteritis exist? A 2-year population-based survey in Nova Scotia, Canada.

BACKGROUND: Febrile gastroenteritis due to Listeria monocytogenes (LM) has been primarily described in foodborne outbreaks. We decided to determine the incidence of sporadic, febrile gastroenteritis due to LM in a large, well-defined North American population over a 2-year period and to compare these cases to sporadic cases of Campylobacter and Salmonella infections occurring concurrently in the community. METHODS: From 1 September 2002 through 31 August 2004, all stool specimens submitted for evaluation of diarrheal illness to a public health laboratory and to a children's hospital serving a population of approximately 350,000 were examined for the presence of Listeria species. Patients identified as having LM in their stool samples were matched with 2 temporally-matched patients with cultures positive for Campylobacter and Salmonella species. Patients with LM and control patients were contacted by telephone, and they answered a questionnaire that examined clinical features and risk factors for diarrheal illness. RESULTS: A total of 7775 stool specimens were submitted during the period 1 September 2002-31 August 2004. Thirty-nine Listeria species were recovered. Seventeen of the species were LM, 13 were Listeria innocua, 3 were Listeria welshimeri, 1 was Listeria grayi, and 4 were other species. Pulsed-field gel electrophoresis results demonstrated no temporal or other clusters, and no seasonality was noted for isolates of LM. Preexisting gastrointestinal problems were much more common in patients with LM (P=.001) than in patients with Campylobacter or Salmonella infections. CONCLUSIONS: Sporadic gastroenteritis due to LM appears to be an uncommon illness, and routine screening of stool samples for LM remains unwarranted. Preexisting gastrointestinal disease may be a risk factor for infection of the gastrointestinal tract with LM.