Pulmonary gene delivery is thought to play an important role in treating genetically related diseases and may induce immunity towards pathogens entering the body via the airways. In this study we prepared poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles bearing polyethyleneimine (PEI) on their surface and characterized them for their potential in serving as non-viral gene carriers to the pulmonary epithelium. Particles that were synthesized at different PLGA-PEI ratios and loaded with DNA in several PEI-DNA ratios, exhibited narrow size distribution in all formulations, with mean particle sizes ranging between 207 and 231 nm. Zeta potential was strongly positive (above 30 mV) for all the PEI-DNA ratios examined and the loading efficiency exceeded 99% for all formulations. Internalization of the DNA-loaded PLGA-PEI nanoparticles was studied in the human airway submucosal epithelial cell line, Calu-3, and DNA was detected in the endo-lysosomal compartment 6 h after particles were applied. Cytotoxicity of these nanoparticles was dependent on the PEI-DNA ratio and best cell viability was achieved by PEI-DNA ratios 1:1 and 0.5:1. These findings demonstrate that PLGA-PEI nanoparticles are a potential new delivery system to carry genes to the lung epithelium.

While immunological memory has long been considered the province of T- and B- lymphocytes, it has recently been reported that innate cell populations are capable of mediating memory responses. We now show that an innate memory immune response is generated in mice following infection with vaccinia virus, a poxvirus for which no cognate germline-encoded receptor has been identified. This immune response results in viral clearance in the absence of classical adaptive T and B lymphocyte populations, and is mediated by a Thy1+ subset of natural killer (NK) cells. We demonstrate that immune protection against infection from a lethal dose of virus can be adoptively transferred with memory hepatic Thy1+ NK cells that were primed with live virus. Our results also indicate that, like classical immunological memory, stronger innate memory responses form in response to priming with live virus than a highly attenuated vector. These results demonstrate that a defined innate memory cell population alone can provide host protection against a lethal systemic infection through viral clearance.

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Candidate HIV-1 vaccine regimens utilizing intramuscularly (i.m.) administered recombinant adenovirus (rAd)-based vectors can induce potent mucosal cellular immunity. However, the degree to which mucosal rAd vaccine routing might alter the quality and anatomic distribution of vaccine-elicited CD8 ؉ T lymphocytes remains unclear. We show that the route of vaccination critically impacts not only the magnitude but also the phenotype and trafficking of antigen-specific CD8 ؉ T lymphocytes in mice. I.m. rAd immunization induced robust local transgene expression and elicited high-frequency, polyfunctional CD8 ؉ T lymphocytes that trafficked broadly to both systemic and mucosal compartments. In contrast, intranasal (i.n.) rAd immunization led to similarly robust local transgene expression but generated low-frequency, monofunctional CD8 ؉ T lymphocytes with restricted anatomic trafficking patterns. Respiratory rAd immunization elicited systemic and mucosal CD8؉ T lymphocytes with phenotypes and trafficking properties distinct from those elicited by i.m. or i.n. rAd immunization. Our findings indicate that the anatomic microenvironment of antigen expression critically impacts the phenotype and trafficking of antigen-specific CD8 ؉ T lymphocytes.Acute human immunodeficiency virus type 1 (HIV-1) infection is accompanied by a massive, irreversible destruction of memory CD4ϩ T lymphocytes, particularly within the intestinal mucosa (11,26,30,42), as a result of the high proportion of effector/memory target cells within the intestinal lamina propria. Chronic HIV-1 infection is characterized by inflammation within the intestinal mucosa, breakdown of epithelialbarrier integrity, and translocation of gut microflora from the intestinal lumen (10, 24). These processes may drive systemic inflammation and contribute to HIV-1 disease progression. Therefore, vaccination strategies that enhance mucosal cellular immunity and attenuate the mucosal immunopathology of HIV-1 infection would be desirable.Recombinant adenovirus (rAd) vectors are potent inducers of cellular immunity (3, 12, 25), and we have recently demonstrated that intramuscular (i.m.) rAd immunization transiently activates peripheral antigen-specific CD8 ϩ T lymphocytes and allows them to migrate to mucosal surfaces and establish potent, durable mucosal cellular immunity (22). Moreover, we have shown that an i.m. delivered heterologous rAd primeboost regimen prevented the destruction of CD4 ϩ T lymphocytes within the intestinal mucosa and attenuated disease progression following simian immunodeficiency virus (SIV) challenge (29). Notably, this vaccine regimen did not contain the SIV Env protein, indicating that cellular mucosal immunity likely played a critical role in abrogating mucosal CD4 ϩ Tlymphocyte destruction.While our laboratory and others have observed potent mucosal CD8 ϩ T-lymphocyte responses after i.m. immunization with rAd vectors (2, 21, 28, 41) and other vaccine modalities (40-41), other studies have suggested that mucosal routing of vaccine vectors may opti...

Objectives To determine the clinical sequelae of long covid for a year after infection in patients with mild disease and to evaluate its association with age, sex, SARS-CoV-2 variants, and vaccination status. Design Retrospective nationwide cohort study. Setting Electronic medical records from an Israeli nationwide healthcare organisation. Population 1 913 234 Maccabi Healthcare Services members of all ages who did a polymerase chain reaction test for SARS-CoV-2 between 1 March 2020 and 1 October 2021. Main outcome measures Risk of an evidence based list of 70 reported long covid outcomes in unvaccinated patients infected with SARS-CoV-2 matched to uninfected people, adjusted for age and sex and stratified by SARS-CoV-2 variants, and risk in patients with a breakthrough SARS-CoV-2 infection compared with unvaccinated infected controls. Risks were compared using hazard ratios and risk differences per 10 000 patients measured during the early (30-180 days) and late (180-360 days) time periods after infection. Results Covid-19 infection was significantly associated with increased risks in early and late periods for anosmia and dysgeusia (hazard ratio 4.59 (95% confidence interval 3.63 to 5.80), risk difference 19.6 (95% confidence interval 16.9 to 22.4) in early period; 2.96 (2.29 to 3.82), 11.0 (8.5 to 13.6) in late period), cognitive impairment (1.85 (1.58 to 2.17), 12.8, (9.6 to 16.1); 1.69 (1.45 to 1.96), 13.3 (9.4 to 17.3)), dyspnoea (1.79 (1.68 to 1.90), 85.7 (76.9 to 94.5); 1.30 (1.22 to 1.38), 35.4 (26.3 to 44.6)), weakness (1.78 (1.69 to 1.88), 108.5, 98.4 to 118.6; 1.30 (1.22 to 1.37), 50.2 (39.4 to 61.1)), and palpitations (1.49 (1.35 to 1.64), 22.1 (16.8 to 27.4); 1.16 (1.05 to 1.27), 8.3 (2.4 to 14.1)) and with significant but lower excess risk for streptococcal tonsillitis and dizziness. Hair loss, chest pain, cough, myalgia, and respiratory disorders were significantly increased only during the early phase. Male and female patients showed minor differences, and children had fewer outcomes than adults during the early phase of covid-19, which mostly resolved in the late period. Findings remained consistent across SARS-CoV-2 variants. Vaccinated patients with a breakthrough SARS-CoV-2 infection had a lower risk for dyspnoea and similar risk for other outcomes compared with unvaccinated infected patients. Conclusions This nationwide study suggests that patients with mild covid-19 are at risk for a small number of health outcomes, most of which are resolved within a year from diagnosis.

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