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.

Vaccination, especially with multiple doses, provides substantial population-level protection against COVID-19, but emerging variants of concern (VOC) and waning immunity represent significant risks at the individual level. Here we identify correlates of protection (COP) in a multicenter prospective study following 607 healthy individuals who received three doses of the Pfizer-BNT162b2 vaccine approximately six months prior to enrollment. We compared 242 individuals who received a fourth dose to 365 who did not. Within 90 days of enrollment, 239 individuals contracted COVID-19, 45% of the 3-dose group and 30% of the four-dose group. The fourth dose elicited a significant rise in antibody binding and neutralizing titers against multiple VOCs reducing the risk of symptomatic infection by 37% [95%CI, 15%-54%]. However, a group of individuals, characterized by low baseline titers of binding antibodies, remained susceptible to infection despite significantly increased neutralizing antibody titers upon boosting. A combination of reduced IgG levels to RBD mutants and reduced VOC-recognizing IgA antibodies represented the strongest COP in both the 3-dose group (HR = 6.34, p = 0.008) and four-dose group (HR = 8.14, p = 0.018). We validated our findings in an independent second cohort. In summary combination IgA and IgG baseline binding antibody levels may identify individuals most at risk from future infections.

The SARS-CoV-2 pandemic led to unprecedented testing demands, causing significant testing delays globally. One strategy used for increasing testing capacity was pooled-testing, using a two-stage technique first introduced during WWII. Here we report the development, validation and clinical application of P-BEST - a single-stage pooled-testing strategy that was approved for clinical use in Israel. P-BEST was clinically evaluated using 3,636 side-by-side tests and was able to correctly detect all positive samples and accurately estimate their Ct value. P-BEST was then used to clinically test 837,138 samples using 270,095 PCR tests - a 3.1 fold reduction in the number of tests. Importantly, P-BEST was also used during the Alpha and Delta waves, when positivity rates exceeded 10%, rendering traditional pooling non-practical. We also describe a tablet-based solution that allows performing manual single-stage pooling in settings where liquid dispensing robots are not available. Our data provides a proof-of-concept for large-scale clinical implementation of single-stage pooled-testing for continuous surveillance of multiple pathogens with reduced test costs, and as an important tool for increasing testing efficiency during pandemic outbreaks.

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.