SARS-CoV-2 innate effector associations and viral load in early nasopharyngeal infection.

COVID-19 causes severe disease with poor outcomes. We tested the hypothesis that early SARS-CoV-2 viral infection disrupts innate immune responses. These changes may be important for understanding subsequent clinical outcomes. We obtained residual nasopharyngeal swab samples from individuals who requested COVID-19 testing for symptoms at drive-through COVID-19 clinical testing sites operated by the University of Utah. We applied multiplex immunoassays, real-time polymerase chain reaction assays and quantitative proteomics to 20 virus-positive and 20 virus-negative samples. ACE-2 transcripts increased with infection (OR =17.4, 95% CI [CI] =4.78-63.8) and increasing viral N1 protein transcript load (OR =1.16, CI =1.10-1.23). Transcripts for two interferons (IFN) were elevated, IFN-λ1 (OR =71, CI =7.07-713) and IFN-λ2 (OR =40.2, CI =3.86-419), and closely associated with viral N1 transcripts (OR =1.35, CI =1.23-1.49 and OR =1.33 CI =1.20-1.47, respectively). Only transcripts for IP-10 were increased among systemic inflammatory cytokines that we examined (OR =131, CI =1.01-2620). We found widespread discrepancies between transcription and translation. IFN proteins were unchanged or decreased in infected samples (IFN-γ OR =0.90 CI =0.33-0.79, IFN-λ2,3 OR =0.60 CI =0.48-0.74) suggesting viral-induced shut-off of host antiviral protein responses. However, proteins for IP-10 (OR =3.74 CI =2.07-6.77) and several interferon-stimulated genes (ISG) increased with viral load (BST-1 OR =25.1, CI =3.33-188; IFIT1 OR =19.5, CI =4.25-89.2; IFIT3 OR =245, CI =15-4020; MX-1 OR =3.33, CI =1.44-7.70). Older age was associated with substantial modifications of some effects. Ambulatory symptomatic patients had an innate immune response with SARS-CoV-2 infection characterized by elevated IFN, proinflammatory cytokine and ISG transcripts, but there is evidence of a viral-induced host shut-off of antiviral responses. Our findings may characterize the disrupted immune landscape common in patients with early disease.

Longitudinal investigation of nasopharyngeal pneumococcal carriage in early childhood: The PATCH birth cohort study.

Streptococcus pneumoniae is a common cause of infectious diseases such as pneumonia and sepsis. Its colonization is thought to be the first step in the development of invasive pneumococcal diseases. This study aimed to investigate pneumococcal colonization patterns in early childhood. A longitudinal birth cohort study was conducted for investigating nasopharyngeal colonized pneumococci at 1, 6, 12, 18, 24, and 36 months of age, particularly focusing on the serotype distribution and antimicrobial susceptibilities. Pneumococcal conjugate vaccine (PCV) effect on nasopharyngeal colonization was also assessed. During 2013-2017, 855 infants were enrolled and a total of 107 isolates were recovered from 95 infants during the first three years of life. In this period, the prevalence of pneumococcal colonization increased, with values ranging from 0.2% (2/834) at 1 month of age to 5.9% (19/323) at 36 months of age. The investigation of serotype revealed that 81.1% (73/90) belonged to the non-PCV13 serotypes-23A, 15A, 15C, and 15B. Moreover, PCV13 serotypes significantly decreased during 2014-2015, when routine PCV13 vaccination was initiated in Taiwan. PCV13 introduction may lead to the reduction in the rates of pneumococcal isolates resistant (R) to penicillin. Under conditional PCV13 vaccination, pneumococcal isolates primarily belonged to non-PCV13 serotypes. This non-PCV13 serotype replacement exhibited lower rates of penicillin R isolates, suggesting that PCV13 administration may reduce the antibiotic-nonsusceptible pneumococcal disease burden and antibiotic use.

Roles of T follicular helper cells in the pathogenesis of adenoidal hypertrophy combined with secretory otitis media.

The aim of this study was to investigate the roles of T follicular helper (Tfh) cells in secretory otitis media (SOM) combined with adenoidal hypertrophy (AH).Patients with AH or AH combined with SOM admitted to the Yancheng No. 1 People's Hospital from December 2012 to December 2014 were included. Fourteen age-matched healthy individuals received physical examinations in the hospital served as control. The venous Tfh was determined using flow cytometry, and CD3 + CD4 + CXCR5 + T lymphocytes were defined as Tfh cells. Serum inflammatory factors including IL-8, IL-1b, IL-6, IL-10, TNF, IL-12p70, IL-21, and IgE were determined using commercial kits.Compared with the AH group, the number of CD4 + CXCR5 + T cells in peripheral blood of the AH combined with SOM group showed significant increase. Statistical differences were noticed in the number of the number of CD4 + CXCR5 + T cells in moderate and severe AH groups compared with that of the control group. Statistical differences were identified in the proportion of CD4 + CXCR5 + T cells in the adenoidal tissues between the AH combined with SOM group and AH group (P < .05). For the CD4 + CXCR5 + T cells in adenoidal tissues, no statistical differences were noticed between the moderate and severe AH groups (P > .05). The number of CD4 + CXCR5 + T cells was positively correlated to the serum IL-21. Nevertheless, no correlation was noticed between CD4 + CXCR5 + T cell and serum IL-8, IL-6, IL-10, and IgE.Tfh is involved in the AH combined with SOM in children. Besides, serum IL-21, IL-8, IL-6, IL-10, and IgE may be involved in the onset of SOM in children.

Colonization-induced protection against invasive pneumococcal disease in mice is independent of CD103 driven adaptive immune responses.

Nasopharyngeal colonization with Streptococcus pneumoniae (the pneumococcus) is known to mount protective adaptive immune responses in rodents and humans. However, the cellular response of the nasopharyngeal compartment to pneumococcal colonization and its importance for the ensuing adaptive immune response is only partially defined. Here we show that nasopharyngeal colonization with S. pneumoniae triggered substantial expansion of both integrin αE (CD103) positive dendritic cells (DC) and T lymphocytes in nasopharynx, nasal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLN) of WT mice. However, nasopharyngeal de-colonization and pneumococcus-specific antibody responses were similar between WT and CD103 KO mice or Batf3 KO mice. Also, naïve WT mice passively immunized with antiserum from previously colonized WT and CD103 KO mice were similarly protected against invasive pneumococcal disease (IPD). In summary, the data show that CD103 is dispensable for pneumococcal colonization-induced adaptive immune responses in mice.

Nasopharyngeal Pneumococcal Carriage in Nigeria: a two-site, population-based survey.

Changes in nasopharyngeal (NP) carriage of vaccine-type (VT) Streptococcus pneumoniae can be used to assess the effectiveness of a pneumococcal conjugate vaccine (PCV10). We conducted a baseline carriage survey in rural (Kumbotso, Kano) and urban (Pakoto, Ogun) Nigeria. In this cross-sectional study, we obtained data on demography, clinical history, risk factors, and took NP swabs for pneumococcal culture. We calculated crude and age-standardised carriage prevalence and used log-binomial regression to assess risk factors for carriage. Among children aged <5 years, 92% (95% CI: 88-95%) and 78% (73-82%), respectively, carried any pneumococcus and 48% and 50%, respectively, carried PCV10 serotypes. In Kumbotso, carriage prevalence was >40% across all ages. The age-standardized prevalence of pneumococcal carriage was 66% in Kumbotso and 40% in Pakoto. The most commonly identified serotypes were 19 F, 6 A and 23 F. Risk factors for carriage were young age, recent rhinorrhoea, cohabitation with ≥2 children aged <5 years, and sharing a bed with ≥2 persons. Pneumococcal carriage prevalence is high in this Nigerian population. Persisting prevalence of VT-carriage in older children and adults suggests that PCV10 introduction in children will not eliminate transmission of vaccine serotypes rapidly. High vaccine coverage will therefore be required to ensure full protection of children.

Streptococcus pneumoniae burden and nasopharyngeal inflammation during acute otitis media.

Streptococcus pneumoniae (Spn) is a common respiratory pathogen and a frequent cause of acute otitis media (AOM) in children. The first step in bacterial pathogenesis of AOM is the establishment of asymptomatic colonization in the nasopharynx. We studied Spn bacterial burden in conjunction with neutrophil recruitment and inflammatory gene transcription and cytokine secretion in samples of nasal wash collected from normal and otitis-prone children during health, viral upper respiratory infection without middle ear involvement (URI) and AOM. We found no significant associations between otitis-prone status and any of the measured parameters. However, Spn bacterial burden was significantly correlated with neutrophil recruitment, transcription of IL-8, TNF-α and SOD2, and secretion of TNF-α. We also found that transcription of IL-8 and TNF-α mRNA by neutrophils was significantly correlated with the secretion of these cytokines into the nasopharynx. We conclude that Spn bacterial burden in the NP is a major determinant of neutrophil recruitment to the NP and activity during URI and AOM, and that neutrophils are contributors to the secretion of IL-8 and TNF-α in the NP when the Spn burden is high.

[SPECIES COMPOSITION OF INFECTIOUS FACTORS THAT CAUSE THE REACTIVE ARTHRITIS DEVELOPMENT AND THEIR EFFECT ON ARGINASE-NO-SYNTHASE REGULATORY SYSTEM OF PERIPHERAL BLOOD LYMPHOCYTES].

The own observations results of urogenital, gastrointestinal and nasopharyngeal infectious factors that cause the development of reactive arthritis (PeA) are being presented. The greatest contribution to the development of this disease make Chlamidia trachomatis (36%), Streptococcus haemolyticus (pyogenes) (19%) and hepatitis viruses B and C (10%). As a result of the research a number of kinetic parameters of arginase and NO-synthase reactions in peripheral blood lymphocytes of patients with reactive arthritis was identified. The authentic increase of arginase activity in 3.3 times and eNO-synthase activity decrease by 1,9 times in peripheral blood lymphocytes of patients with PeA, compared to practically healthy donors were determined. Increased activity of arginase and iNO-synthase of lymphocytes indicates changes in immune cells functional activity, which may be due to impaired metabolic and regulatory processes in these cells caused by a bacterial or viral infection.

[Specific clinical and immunological features of chronic diseases of the nasal-associated lymphoid tissue in the children].

The objective of the present work was to study the structure, clinical, and immunological features of various etiological variants of chronic diseases of the nasal-associated lymphoid tissue in the children. A total of 142 children at the age from 3 to 7 years presenting with this pathological condition were available for the observation. The study revealed differences in the clinical course of the disease and the cytokine response (IL-6, Ril-6, TNF, sYNFR55, sTNFR75) at the local and systemic levels for different pathogens (S. aureus, S. pneumoniae, S. pyrogenes).

Association of Streptococcus pneumoniae common protein antigen (CPA) antibodies and pneumococcal nasopharyngeal colonization in HIV-infected and HIV-uninfected African children.

Due to the high cost and limited serotype coverage of pneumococcal conjugate vaccines (PCV), pneumococcal common protein antigens (CPAs) are being investigated as potential vaccine candidates. CPAs are likely to be immunogenic in infants and could confer serotype-independent protection. There are limited data on natural antibody kinetics against CPAs in African populations. We aimed to determine the prevalence of naturally acquired antibody titres to 15 CPAs and explore their association to concurrent pneumococcal nasopharyngeal colonization in children aged 4-7 years with and without underlying HIV-infection and/or previous PCV-vaccination. A 15-plex Luminex assay was established to measure serum IgG titres against "cell-wall associated or surface-exposed" proteins (PspA, PspC, LytB, IgA1-proteinase, SP0082, PdB and PcsB), "membrane-associated" proteins (PsaA, SP0609, SP0749, PpmA, SlrA, StkP and SP2194) as well as the hypothetical protein, SP2027. Archived serum samples from HIV-uninfected (n=212) and HIV-infected (n=74) children were analyzed. Concurrent pneumococcal nasopharyngeal colonization was determined with standard microbiological methods. HIV-uninfected children had significantly higher antibody titres against PspA, PspC, PdB, SP0082, LytB, IgA1 proteinase and PcsB compared to HIV-infected children. In contrast, antibody titres against membrane associated proteins (PsaA, SP2027, PpmA and SlrA) were significantly lower in HIV-uninfected compared to HIV-infected children. Higher antibody titres against PdB, and PcsB were associated with the absence of pneumococcal colonization. There was no association between anti-CPA titres and PCV vaccination. In conclusion PdB and PcsB antigens are potential vaccine-candidates which may protect against pneumococcal colonization and consequently pneumococcal disease.

Protection against pneumococcal infection elicited by immunization with multiple pneumococcal heat shock proteins.

Heat shock proteins (HSPs) play important roles in the pathogenesis of pneumococcal infection, and they are considered as potential protein vaccine antigens. In this study, we investigated the efficacy of immunization with pneumococcal HSPs, including ClpP (hsp100/Clp peptidase subunit), DnaJ (hsp40) and GroEL (hsp60), to protect against pneumococcal carriage, lung colonization and sepsis in mouse models using different serotypes of Streptococcus pneumoniae. In a nasopharyngeal colonization model by serotype 6B or 14 and in a lung colonization model by serotype 19F, immunization with pneumococcal HSPs could elicit effective protection. Likewise, vaccination with ClpP, DnaJ or GroEL allowed significantly longer mouse survival times after lethal intranasal challenge with serotype pneumococcal 2, 3 or 4. Interestingly, combinations of these HSPs could consistently enhance the protection against nasopharynx carriage, lung colonization as well as invasive infection caused by different pneumococcal serotypes. In an in vitro killing assay, anti-sera against ClpP, DnaJ or GroEL could kill S. pneumoniae by polymorphonuclear leukocytes in a complement-dependent way, and combinations of multiple anti-sera against these HSPs could increase the killing ability compared with single anti-sera. Finally, passive immunization studies with anti-sera against pneumococcal HSPs also demonstrated that an additive effect could be achieved by using multiple anti-sera when compared with single anti-sera. Thus, inclusion of multiple pneumococcal HSPs is important for the development of protein-based pneumococcal vaccines.

Immunization with DnaJ (hsp40) could elicit protection against nasopharyngeal colonization and invasive infection caused by different strains of Streptococcus pneumoniae.

Increasing mortality, morbidity and economic costs have been paid to pneumococcal diseases every year. Currently, vaccination is the most promising strategy to reduce the occurrence of pneumococcal infection. In this study, we investigated the protective efficacy of immunization with recombinant DnaJ (hsp40) protein against infections of different serotypes of Streptococcus pneumoniae. We demonstrated that mucosal immunization with DnaJ antigen could induce both systemic and mucosal antibodies for DnaJ and stimulate the release of high levels of IL-10, IFN-γ and IL-17A. Moreover, this mucosal vaccination could reduce nasal or lung colonization of pneumococcus and elicit protection against different serotypes of invasive pneumococcal infections. As well, we found that intraperitoneal immunization with DnaJ could also protect against invasive infections caused by different serotypes of pneumococcus, and passive immunization with antibodies specific for DnaJ confirmed that this protection was antibody-mediated. Our results therefore support the potential of DnaJ as a conserved pneumococcal protein vaccine.

Bacterial toxins and Multiple Sclerosis.

The primary pathogenetic mechanism responsible for the distinctive demyelinating lesions in the Central Nervous System (CNS) in Multiple Sclerosis (MS), first described in remarkable detail by Charcot more than 170 years ago, remains one of the most baffling conundrums in medicine. A possible role for bacterial cell molecules and transportable proteins in the pathogenesis of MS is reviewed. The ability of bacterial toxins to distort immunity and to cause distinctive toxic damage in the nervous system is discussed in the light of largely forgotten data linking bacterial nasopharyngeal infections with optic neuritis, optochiasmatic arachnoiditis and MS. While the blood-brain barrier substantially protects the CNS from hematogenous toxins, there is a route by which the barrier may be by-passed. Data is reviewed which shows that the CSF and extra-cellular fluid circulation is bi-directionally linked to the lymphatic drainage channels of the nasopharyngeal mucosa. While this provides a facility by which the CNS may mount immunological responses to antigenic challenges from within, it is also a route by which products of nasopharyngeal infection may drain into the CNS and be processed by the immune cells of the meninges and Virchow-Robin perivascular spaces. If potentially toxic bacterial products are identified in early MS tissues at these sites, this would provide an entirely new insight into the pathogenetic mechanisms of this frustratingly enigmatic disease.

Nasal vaccination with CpG oligodeoxynucleotide induces protective immunity against non-typeable Haemophilus influenzae in the nasopharynx.

OBJECTIVES: Nasal vaccination is an effective therapeutic regimen for preventing otitis media. Since cholera toxin (CT) is toxic, an alternative adjuvant is required for the development of a nasal vaccine. The efficacy of CpG oligodeoxynucleotide (ODN) as a mucosal adjuvant was examined. METHODS: Mice were immunized intranasally with P6 protein of non-typeable Haemophilus influenzae (NTHi) and adjuvant, CT, or CpG ODN, and P6-specific antibody responses were examined. The expression of P6-specific cytokine mRNA in splenic CD4 T cells was also determined. In addition, NTHi challenges were performed and the NTHi was quantified in nasal washes. RESULTS: P6-specific IgA in nasal wash and serum IgG titers were elevated significantly after nasal immunization. The IgG1/IgG2a ratio in serum from P6+CpG-immunized mice was less than that of P6+CT-immunized mice. Although IL-6 was expression similarly in both groups, IFN-gamma expression was greater in P6+CpG-immunized mice than in P6+CT-immunized mice. Enhanced clearance of NTHi from the nasopharynx was also shown equally in both groups. CONCLUSION: These results indicate that CpG ODN might be an effective mucosal adjuvant, acting by mechanisms that are different from CT. These findings suggest that nasal vaccination with P6 and CpG ODN might be an effective regimen for the induction of NTHi-specific protective immunity.

Antibody-independent, interleukin-17A-mediated, cross-serotype immunity to pneumococci in mice immunized intranasally with the cell wall polysaccharide.

Serotype-specific immunity to Streptococcus pneumoniae is conferred by antibodies to the capsular polysaccharides, which define the 90 known serotypes. Whether antibody to the species-common cell wall polysaccharide (C-Ps) is protective has been a matter of controversy. Here we show that C-Ps given intranasally with mucosal adjuvant increased the resistance of mice to experimental nasopharyngeal colonization by capsulated S. pneumoniae of serotype 6B. This immunity could be induced in mice congenitally lacking immunoglobulin but was dependent upon CD4+ T cells. Elimination of the charged amino group on the polymer backbone by N acetylation of C-Ps reduced the immunity, as did treatment of the mice with antibody to the cytokine interleukin-17A at the time of challenge, both consistent with the hypothesis of T-cell activation due to the zwitterionic motif of the polymer. C-Ps also protected in a model of fatal aspiration pneumonia by heavily capsulated serotype 3. These findings suggest a novel immunization strategy against S. pneumoniae.

PppA, a surface-exposed protein of Streptococcus pneumoniae, elicits cross-reactive antibodies that reduce colonization in a murine intranasal immunization and challenge model.

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an approximately 20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.

Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant.

Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective.

Multiserotype protection of mice against pneumococcal colonization of the nasopharynx and middle ear by killed nonencapsulated cells given intranasally with a nontoxic adjuvant

Intranasal challenge of C57BL/6 mice with Streptococcus pneumoniae serotypes 6B, 14, and 23F produced colonization of the middle ear and NP. Intranasal vaccination with ethanol-killed nonencapsulated cells with adjuvant protected both sites. Of four nontoxic adjuvants tested, the cholera toxin B subunit was most effective and least nonspecifically protective

Effects of large dosages of amoxicillin/clavulanate or azithromycin on nasopharyngeal carriage of Streptococcus pneumoniae, Haemophilus influenzae, nonpneumococcal alpha-hemolytic streptococci, and Staphylococcus aureus in children with acute otitis media.

Prior use of antibiotics is associated with carriage of resistant bacteria. Colonization by Streptococcus pneumoniae, Haemophilus influenzae, nonpneumococcal alpha-hemolytic streptococci (NPAHS), and Staphylococcus aureus was evaluated in children receiving antibiotic therapy for acute otitis media and in untreated, healthy control subjects. Children were randomly assigned to receive either amoxicillin/clavulanate (90 mg/kg per day) or azithromycin. Swabs were obtained before initiating therapy and again 2 weeks and 2 months after initiating therapy. We also obtained swabs from control subjects at the time of enrollment and 2 weeks and 2 months after enrollment. The decrease in the rate of carriage of S. pneumoniae and H. influenzae at 2 weeks was significant only in the amoxicillin/clavulanate group (P<.001 and P=.005, respectively). The rate of nasopharyngeal colonization with NPAHS among treated patients increased from 23% to 39% at 2 months (P=.01). This increase was similar for both treatment groups. These results suggest that the competitive balance between organisms is altered by antibiotic therapy.