The COVID-19 Pandemic as an Opportunity for Unravelling the Causative Association between Respiratory Viruses and Pneumococcus-Associated Disease in Young Children: A Prospective Cohort Study (preprint)

BACKGROUNDIn young children, rates of community-acquired alveolar pneumonia (CAAP) or invasive pneumococcal disease (IPD) have been associated with respiratory syncytial virus (RSV), human metapneumovirus (hMPV), influenza (flu), and parainfluenza (PIV) (collectively termed here as pneumococcal disease-associated viruses [PDA-viruses]). However, their contribution to the pathogenesis of pneumococcal-associated disease has not yet been elucidated. The COVID-19 pandemic provided a unique opportunity to examine the question. METHODSThis prospective study comprised all children <5 years, living in southern Israel, during 2016 through 2021. The data were derived from multiple ongoing prospective cohort surveillance programs and include: hospital visits for CAAP, non-CAAP lower respiratory infections (LRI); nasopharyngeal pneumococcal carriage (<3 years of age); respiratory virus activity; all-ages COVID-19 episodes; and IPD in children <5 years (nationwide) A hierarchical negative binominal regression model was developed to estimate the proportion of the disease outcomes attributable to each of the viruses from monthly time series data, stratified by age and ethnicity. A separate model was fit for each outcome, with covariates that included a linear time trend, 12-month harmonic variables to capture unexplained seasonal variations, and the proportion of tests positive for each virus in that month. FINDINGSDuring 2016 through 2021, 3,204, 26,695, 257, and 619 episodes of CAAP, non-CAAP LRI, pneumococcal bacteremic pneumonia and non-pneumonia IPD, respectively, were reported. Compared to 2016-2019, broad declines in the disease outcomes were observed shortly after the pandemic surge, coincident with a complete disappearance of all PDA-viruses and continued circulation of rhinovirus (RhV) and adenovirus (AdV). From April 2021, off-season and abrupt surges of all disease outcomes occurred, associated with similar dynamics among the PDA-viruses, which re-emerged sequentially. Using our model fit to the entire 2016-2021 period, 82% (95% CI, 75-88%) of CAAP episodes in 2021 were attributable to the common respiratory viruses, as were 22%-31% of the other disease outcomes. Virus-specific contributions to CAAP were: RSV, 49% (95% CI, 43-55%); hMPV, 13% (10-17%); PIV, 11% (7-15%); flu, 7% (1-13%). RhV and AdV did not contribute. RSV was the main contributor in all outcomes, especially in infants. Pneumococcal carriage prevalence remained largely stable throughout the study. INTERPRETATIONRSV and hMPV play a critical role in the burden of CAAP and pneumococcal disease in children. Interventions targeting these viruses could have a secondary effect on the disease burden typically attributed to bacteria. Research in ContextO_ST_ABSEvidence before this studyC_ST_ABSCommunity-acquired alveolar pneumonia (CAAP) and invasive pneumococcal disease (IPD) in young children have often been associated with specific respiratory viruses, namely respiratory syncytial virus (RSV) human metapneumovirus (hMPV) influenza viruses (flu), and parainfluenza viruses (PIV) (termed in the current article pneumococcal disease-associated viruses [PDA-viruses]). However, their causative role as co-pathogens has not yet been fully elucidated. Pneumococcal conjugate vaccines (PCVs) significantly reduce hospitalization for viral lower respiratory infections (LRIs), suggesting that viral-pneumococcal coinfections are common and play a role in the pathogenesis of pneumococcal respiratory infections. However, in theory, the strongest demonstration of the causative role of respiratory viruses on pneumococcus-associated diseases would derive from measuring the impact of elimination of one or more of the respiratory viruses during the expected respiratory season. Shortly after the start of the COVID-19 pandemic, multiple reports have demonstrated reduced IPD and LRI rates among young children, coincident with dramatically reduced rates of the PDA-viruses globally. Initially, the reduced pneumococcal disease rates were attributed to non-pharmaceutical interventions that might reduce pneumococcal transmission in the community. However, continuous, virtually unchanged pneumococcal carriage rates were reported in multiple studies, strongly suggesting the reduced circulation of S. pneumoniae was not significantly contributing to disease reduction. Surprisingly, pneumococcus-associated diseases and PDA-viruses simultaneously re-emerged in 2021 during the off-season. In contrast to PDA-viruses, other viruses, such as adenovirus and rhinovirus did not show any of the patterns discussed above. We searched PubMed on June 1st, 2022, for studies since 2012 using the following terms: ("COVID-19" or "SARS-Cov-2") and ("S. pneumoniae" or "pneumococcus" or "IPD" or "respiratory virus" or respiratory syncytial virus" or "hMPV" or "influenza" or "parainfluenza" or "adenovirus" or "rhinovirus" or "lower respiratory infection"). The search was for English literature and unrestricted by date. Added value of this studyThree unique characteristics of the COVID-19 pandemic-induced abnormal dynamics, coupled with multiple ongoing cohort studies in young children, contributed to the historic opportunity to model and quantify the attributable role of the various common respiratory viruses to four pneumococcus-associated disease outcomes (CAAP, non-CAAP LRIs, pneumococcal bacteremic pneumonia and non-pneumonia IPD): First, the full seasonal disappearance of all PDA-viruses shortly after the start of the pandemic, in the presence of continuous, uninterrupted pneumococcal carriage and continuous unchanged rhinovirus and adenovirus activity. Second, the off-season resurgence of the PDA-viruses in 2021. Third, the sequential, rather than simultaneous, re-emergence of the PDA-viruses. The analysis in this study suggests that several of the respiratory viruses, particularly RSV and hMPV, play an important causative role in the pathogenesis of pneumococcal diseases and related conditions. Furthermore, the proportion attributable to each of the PDA-viruses for each of the four studied disease outcomes, and each of the age groups (<1, 1, and 2-4 years of age) could be demonstrated. Implication of all the available findingsOur findings add evidence about the absolute and relative contribution of common respiratory viruses to the burden of pneumococcal diseases and related conditions in young children, likely to be caused, at least in part, by virus-pneumococcus interaction or coinfections. The strong predominance of RSV contribution compared to other viruses in all studied disease outcomes suggests that interventions that target viruses could have secondary effects on the burden of diseases typically attributed to bacteria.

Correlates of protection for booster doses of the BNT162b2 vaccine (preprint)

Variants of concern (VOC) of SARS-CoV2 and waning immunity pose a serious global problem. Overall, vaccination and prior infection provide significant protection, but some individuals remain susceptible to infection and severe disease. Rigorously identifying correlates of protection (COP) is necessary to identify these susceptible populations. We conducted a multicenter prospective study assessing the association between serological profiles and the risk for SARS-CoV-2 infection, comparing those vaccinated with three to four doses of Pfizer BNT162b2 vaccine. We identified several IgG and IgA binding markers that were COPs. The strongest COP was reduced IgG levels to RBD mutants and IgA levels to VOCs (three-dose-group HR=6.34, p=0.008; four-dose-group HR=8.14, p=0.018). Most importantly, we identified a subset of vaccinated individuals with low antibody levels that generated a significant boost in neutralizing antibody titers after a fourth dose, but were still at significantly increased susceptibility to infection.

Correlates of protection for booster doses of the BNT162b2 vaccine (preprint)

Variants of concern (VOC) of SARS-CoV2 and waning immunity pose a serious global problem. Herein, we aimed to identify novel correlates of protection (COPs) against symptomatic SARS-CoV-2 infection. We conducted a Multicenter prospective study assessing the association between serological profiles and the risk for SARS-CoV-2 infection, comparing those vaccinated with three to four doses of Pfizer BNT162b2 vaccine. Of 608 healthy adults, 365 received three doses and 243 received four doses. During the first 90 days of followup, 239 (39%) were infected, of whom 165/365 (45%) received 3 doses and 74/243 (30%) four doses. We found that the fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple variants, and reduced the risk of symptomatic infection by 37% [95% I, 15% - 54%]. We found several binding IgG and IgA markers and their combinations that were COPs. The strongest association with infection risk was IgG levels to RBD mutants and IgA levels to VOCs, which was a COP in the three-dose group (HR=6.34, p=0.008) and in the four-dose group (HR=8.14, p=0.018). A combination of two commercially available ELISA assays were also associated with protection in both groups (HR = 1.84, p = 0.002; HR = 2.01, p = 0.025, respectively). In a subset, comparing those with low to high antibody levels before 4th dose, despite a significant rise in neutralizing antibody titers against both omicron variants, the number of infections in the low group (n=16) was significantly higher than in the high group (n=7, 43% vs. 20%, p=0.051). We demonstrated that following immunization with three or four vaccine doses, combinations of IgA and IgG levels are associated with protection from symptomatic infection. In addition, we identified a subpopulation of healthy adult individuals with low-baseline levels of antibodies after 3 doses which are at an increased risk for SARS-CoV-2 infection despite receiving a fourth dose. These findings warrant further study of this group, assessing whether they are at a higher risk for developing severe disease or may spread infection more readily than others.

Covid-19 Vaccine Effectiveness in Healthcare Personnel in six Israeli Hospitals (CoVEHPI) (preprint)

BackgroundMethodologically rigorous studies on Covid-19 vaccine effectiveness (VE) in preventing SARS-CoV-2 infection are critically needed to inform national and global policy on Covid-19 vaccine use. In Israel, healthcare personnel (HCP) were initially prioritized for Covid-19 vaccination, creating an ideal setting to evaluate real-world VE in a closely monitored population. MethodsWe conducted a prospective study among HCP in 6 hospitals to estimate the effectiveness of the BNT162b2 mRNA Covid-19 vaccine in preventing SARS-CoV-2 infection. Participants filled out weekly symptom questionnaires, provided weekly nasal specimens, and three serology samples - at enrollment, 30 days and 90 days. We estimated VE against PCR-confirmed SARS-CoV-2 infection using the Cox Proportional Hazards model and against a combined PCR/serology endpoint using Fishers exact test. FindingsOf the 1,567 HCP enrolled between December 27, 2020 and February 15, 2021, 1,250 previously uninfected participants were included in the primary analysis; 998 (79.8%) were vaccinated with their first dose prior to or at enrollment, all with Pfizer BNT162b2 mRNA vaccine. There were four PCR-positive events among vaccinated participants, and nine among unvaccinated participants. Adjusted two-dose VE against any PCR- confirmed infection was 94.5% (95% CI: 82.6%-98.2%); adjusted two-dose VE against a combined endpoint of PCR and seroconversion for a 60-day follow-up period was 94.5% (95% CI: 63.0%-99.0%). Five PCR-positive samples from study participants were sequenced; all were alpha variant. InterpretationOur prospective VE study of HCP in Israel with rigorous weekly surveillance found very high VE for two doses of Pfizer BNT162b2 mRNA vaccine against SARS-CoV-2 during a period of predominant alpha variant circulation. FundingClalit Health Services

Decline in pneumococcal disease in young children during the COVID-19 pandemic associated with suppression of seasonal respiratory viruses, despite persistent pneumococcal carriage: A prospective cohort study (preprint)

Background: Invasive pneumococcal disease (IPD) declined during the COVID-19 pandemic. Previous studies hypothesized that this was due to reduced pneumococcal transmission resulting from non-pharmacological interventions. We used multiple ongoing cohort surveillance projects in children <5 years to test this hypothesis. Methods: The first SARS-CoV-2 cases were detected in February-2020, resulting in a full lockdown, followed by several partial restrictions. Data from ongoing surveillance projects captured the incidence dynamics of community-acquired alveolar pneumonia (CAAP), non-alveolar lower respiratory infections necessitating chest X-rays (NA-LRI), nasopharyngeal pneumococcal carriage in non-respiratory visits, nasopharyngeal respiratory virus detection (by PCR), and nationwide invasive pneumococcal disease (IPD). Monthly rates (January-2020 through February-2021 vs. mean monthly rates 2016-2019 [expected rates]) adjusted for age and ethnicity, were compared. Findings: CAAP and bacteremic pneumococcal pneumonia were strongly reduced (incidence rate ratios, [IRRs] 0.07 and 0.19, respectively); NA-LRI and non-pneumonia IPD were also reduced, with a lesser magnitude (IRRs, 0.46 and 0.42, respectively). In contrast, pneumococcal carriage prevalence was only slightly reduced and density of colonization and pneumococcal serotype distributions were similar to previous years. The pneumococcus-associated disease decline was temporally associated with a full suppression of RSV, influenza viruses, and hMPV, often implicated as co-pathogens with pneumococcus. In contrast, adenovirus, rhinovirus, and parainfluenza activities were within or above expected levels. Interpretation: Reductions in pneumococcal and pneumococcus-associated diseases occurring during the COVID-19 pandemic were not predominantly related to reduced pneumococcal transmission and carriage but were strongly associated with the complete disappearance of specific respiratory viruses. Funding: Partially funded by Pfizer, Inc.

Evidence for increased breakthrough rates of SARS-CoV-2 variants of concern in BNT162b2 mRNA vaccinated individuals (preprint)

The SARS-CoV-2 pandemic has been raging for over a year, creating global detrimental impact. The BNT162b2 mRNA vaccine has demonstrated high protection levels, yet apprehension exists that several variants of concerns (VOCs) can surmount the immune defenses generated by the vaccines. Neutralization assays have revealed some reduction in neutralization of VOCs B.1.1.7 and B.1.351, but the relevance of these assays in real life remains unclear. Here, we performed a case-control study that examined whether BNT162b2 vaccinees with documented SARS-CoV-2 infection were more likely to become infected with B.1.1.7 or B.1.351 compared with unvaccinated individuals. Vaccinees infected at least a week after the second dose were disproportionally infected with B.1.351 (odds ratio of 8:1). Those infected between two weeks after the first dose and one week after the second dose, were disproportionally infected by B.1.1.7 (odds ratio of 26:10), suggesting reduced vaccine effectiveness against both VOCs under different dosage/timing conditions. Nevertheless, the B.1.351 incidence in Israel to-date remains low and vaccine effectiveness remains high against B.1.1.7, among those fully vaccinated. These results overall suggest that vaccine breakthrough infection is more frequent with both VOCs, yet a combination of mass-vaccination with two doses coupled with non-pharmaceutical interventions control and contain their spread.

SARS CoV-2 escape variants exhibit differential infectivity and neutralization sensitivity to convalescent or post-vaccination sera (preprint)

Towards eradicating COVID19, developing vaccines that induce high levels of neutralizing antibodies is a main goal. As counter measurements, viral escape mutants rapidly emerge and potentially compromise vaccine efficiency. Herein we monitored ability of convalescent or Pfizer-BTN162b2 post-vaccination sera to neutralize wide-type SARS-CoV2 or its UK-B.1.1.7 and SA-B.1.351 variants. Relative to convalescent sera, post-vaccination sera exhibited higher levels of neutralizing antibodies against wild-type or mutated viruses. However, while SARS-CoV2 wild-type and UK-N501Y were similarly neutralized by tested sera, the SA-N501Y/K417N/E484K variant moderately escaped neutralization. Significant contribution to infectivity and sensitivity to neutralization was attributed to each of the variants and their single or combined mutations, highlighting alternative mechanisms by which prevalent variants with either N501Y or E484K/K417N mutations spread. Our study validates the clinical significance of currently administered vaccines, but emphasizes that their efficacy may be compromised by circulated variants, urging the development of new ones with broader neutralization functions.

Full genome viral sequences inform patterns of SARS-CoV-2 spread into and within Israel (preprint)

Full genome sequences are increasingly used to track the geographic spread and transmission dynamics of viral pathogens. Here, with a focus on Israel, we sequenced 212 SARS-CoV-2 sequences and use them to perform a comprehensive analysis to trace the origins and spread of the virus. A phylogenetic analysis including thousands of globally sampled sequences allowed us to infer multiple independent introductions into Israel, followed by local transmission. Returning travelers from the U.S. contributed dramatically more to viral spread relative to their proportion in incoming infected travelers. Using phylodynamic analysis, we estimated that the basic reproduction number of the virus was initially around ~2.0-2.6, dropping by two-thirds following the implementation of social distancing measures. A comparison between reported and model-estimated case numbers indicated high levels of transmission heterogeneity in SARS-CoV-2 spread, with between 1-10% of infected individuals resulting in 80% of secondary infections. Overall, our findings underscore the ability of this virus to efficiently transmit between and within countries, as well as demonstrate the effectiveness of social distancing measures for reducing its spread.

Efficient high throughput SARS-CoV-2 testing to detect asymptomatic carriers (preprint)

The COVID-19 pandemic is rapidly spreading throughout the world. Recent reports suggest that 10-30% of SARS-CoV-2 infected patients are asymptomatic. Other studies report that some subjects have significant viral shedding prior to symptom onset. Since both asymptomatic and pre-symptomatic subjects can spread the disease, identifying such individuals is critical for effective control of the SARS-CoV-2 pandemic. Therefore, there is an urgent need to increase diagnostic testing capabilities in order to also screen asymptomatic carriers. In fact, such tests will be routinely required until a vaccine is developed. Yet, a major bottleneck of managing the COVID-19 pandemic in many countries is diagnostic testing, due to limited laboratory capabilities as well as limited access to genome-extraction and Polymerase Chain Reaction (PCR) reagents. We developed P-BEST - a method for Pooling-Based Efficient SARS-CoV-2 Testing, using a non-adaptive group-testing approach, which significantly reduces the number of tests required to identify all positive subjects within a large set of samples. Instead of testing each sample separately, samples are pooled into groups and each pool is tested for SARS-CoV-2 using the standard clinically approved PCR-based diagnostic assay. Each sample is part of multiple pools, using a combinatorial pooling strategy based on compressed sensing designed for maximizing the ability to identify all positive individuals. We evaluated P-BEST using leftover samples that were previously clinically tested for COVID-19. In our current proof-of-concept study we pooled 384 patient samples into 48 pools providing an 8-fold increase in testing efficiency. Five sets of 384 samples, containing 1-5 positive carriers were screened and all positive carriers in each set were correctly identified. P-BEST provides an efficient and easy-to-implement solution for increasing testing capacity that will work with any clinically approved genome-extraction and PCR-based diagnostic methodologies.