Background: Healthcare workers (HCWs) are at high risk of breakthrough SARS-CoV-2 infections despite complete vaccination schedules. There are gaps in our understanding of the specific antibody isotypes and cytokine profiles produced during an infection following vaccination. In this study, we evaluated SARS-CoV-2⁻specific antibody isotypes and their association with cytokine production in HCWs with breakthrough infections. Methods: Serum samples from 114 HCWs were analyzed for antibody isotypes against the nucleoprotein (NCP) and the receptor binding domain (RBD) of the spike protein, as well as for a panel of 13 cytokines. Results: Vaccinated SARS-CoV-2⁺ HCWs showed a higher prevalence of anti-SARS-CoV-2 antibodies against NCP (IgM = 93.8%, IgG = 93.8%, IgA = 28.1%) and RBD (IgM = 46.9%, IgG = 100%, IgA = 90.6%). A specific IgM response to NCP was more frequent in vaccinated SARS-CoV-2⁺ individuals, whereas IgA responses were predominantly specific for RBD. Both pro- and anti-inflammatory cytokines were elevated in vaccinated HCWs with breakthrough infections compared with unvaccinated and uninfected individuals. Interestingly, infected IgG+ HCWs with IgM specific for both NCP and RBD exhibited significantly higher IL-8, IL-6, TNF-α, IFN-γ, IL-2, IL-10, and TGF-β concentrations. Conclusion. Our data show that breakthrough infections in vaccinated HCWs induce a robust pro-and anti-inflammatory cytokine profile, which is associated with a broader IgM response directed against both NCP and RBD.

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the start, the growth curve, total protein concentration and colony-forming units (CFUs) of each strain were measured. From each co-culture, CFUs and total RNA were obtained, and transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media, showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFUs recovered from the Hp ΔvapD strain from AGS cells decreased after 36 h. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt, to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells.

Helicobacter pylori can persist intracellularly, offering protection against the immune system and antimicrobial treatments, which promote chronic infection. Previous studies revealed that the virulence-associated protein D ( vapD ) gene is transcriptionally induced during H. pylori intracellular infection and was associated with bacterial persistence, as deletion of vapD impairs bacterial intracellular survival. However, whether VapD protein is expressed and localized during intracellular infection had not been demonstrated. The aim of this study was to detect the VapD protein when H. pylori is inside eukaryotic cells. Polyclonal antibodies against VapD and immunofluorescence microscopy were used to detect the VapD expression in co-cultures of H. pylori strain 26695 and AGS cells. The H. pylori strain Tx30a, which lacks the vapD gene, was used as a negative control. VapD expression was detected exclusively in AGS cells infected with strain 26695. Confocal microscopy confirmed the intracellular localization of the VapD signal, which coincided with the localization of H. pylori within AGS cells. In addition, a high proportion of H. pylori -infected AGS cells were positive for VapD signal. Together, these results provide first direct evidence of VapD expression during H. pylori intracellular infection and extend previous transcriptional and genetic studies by confirming VapD production at the protein level, supporting its association with the intracellular state of H. pylori .

The Helicobacter pylori vapD gene is transcribed and expressed when the bacteria are within the gastric cell. In this current study, we investigated how vapD knockout affects the survival of H. pylori inside human gastric adenocarcinoma cells. We constructed an H. pylori 26695 vapD (Hp ΔvapD) mutant strain. H. pylori 26695 wt and Hp ΔvapD strains were grown in synthetic media and were co-cultured with AGS cells. From the first, the growth curve, total protein concentration and colony-forming units (CFU) of each strain were measured. From each co-culture, CFU and total RNA were obtained, transcript levels of GAPDH, vapD, vacA, ureA, and 16s Hp were measured by qRT-PCR. Hp ΔvapD did not affect the growth rate of the strain in synthetic media showing that the vapD gene is not necessary when the bacteria grow outside eukaryote cells. However, in the intracellular environment, the number of CFU recovered from the Hp ΔvapD strain from AGS cells decreases after 36 hours. Transcription levels of the vacA gene from the Hp ΔvapD strain were 10,000-fold lower than those of H. pylori wt to the point of being undetectable. The results suggest that the vapD gene contributed to maintaining H. pylori inside gastric cells.