Increasing human monoclonal antibody cloning efficiency with a whole-cell modified immunoglobulin-capture assay (mICA).

Expression cloning of fully human monoclonal antibodies (hmAbs) is seeing powerful utility in the field of vaccinology, especially for elucidating vaccine-induced B-cell responses and novel vaccine candidate antigen discovery. Precision of the hmAb cloning process relies on efficient isolation of hmAb-producing plasmablasts of interest. Previously, a novel immunoglobulin-capture assay (ICA) was developed, using single protein vaccine antigens, to enhance the pathogen-specific hmAb cloning output. Here, we report a novel modification of this single-antigen ICA using formalin-treated, fluorescently stained whole cell suspensions of the human bacterial invasive pathogens, Streptococcus pneumoniae and Neisseria meningitidis. Sequestration of IgG secreted by individual vaccine antigen-specific plasmablasts was achieved by the formation of an anti-CD45-streptavidin and biotin anti-IgG scaffold. Suspensions containing heterologous pneumococcal and meningococcal strains were then used to enrich for polysaccharide- and protein antigen-specific plasmablasts, respectively, during single cell sorting. Following application of the modified whole-cell ICA (mICA), ~61% (19/31) of anti-pneumococcal polysaccharide hmAbs were cloned compared to 14% (8/59) obtained using standard (non-mICA) methods - representing a ~4.4-fold increase in hmAb cloning precision. A more modest ~1.7-fold difference was obtained for anti-meningococcal vaccine hmAb cloning; ~88% of hmAbs cloned via mICA versus ~53% cloned via the standard method were specific for a meningococcal surface protein. VDJ sequencing revealed that cloned hmAbs reflected an anamnestic response to both pneumococcal and meningococcal vaccines; diversification within hmAb clones occurred by positive selection for replacement mutations. Thus, we have shown successful utilization of whole bacterial cells in the ICA protocol enabling isolation of hmAbs targeting multiple disparate epitopes, thereby increasing the power of approaches such as reverse vaccinology 2.0 (RV 2.0) for bacterial vaccine antigen discovery.

Mesenteric lipoma with small bowel volvulus: A rare cause of upper gastrointestinal obstruction (a case report and literature review).

INTRODUCTION: Small bowel volvulus due to mesenteric lipoma is a rare clinical entity. It poses both a diagnostic and therapeutic challenge. Small bowel mesenteric lipoma is a rare cause of small bowel obstruction. We present the case of a patient admitted to our emergency department for a small bowel volvulus due to a mesenteric lipoma with small intestine obstruction. PATIENT AND METHOD: A 61 years old man, with diabetes since 25 years with antidiabetics oral medication, vaccinated against Covid 19 (two doses) who presented with peri-umbilical pain for two months, constipation and melaena, complicated 3 days before his admission by obstructive symptoms and vomiting with apyrexia and overall health state alteration. The physical examination noticed abdomen distension and the abdominal CT scan revealed a large fatty mass of the hypochondrium and left flank, roughly oval with regular borders, well limited measuring 124 × 86 mm of height of 126 mm thought to be a liposarcoma. The patient underwent enbloc resection of 20 cm of small bowel with the mass and end to end anastomosis of the ileo-ileum. The postoperative course was uneventful and he was been discharged from hospital on day 5. DISCUSSION: Mesenteric lipomas are diagnosed incidentally after laparoscopy or laparatomy. Ultrasound shows a well defined homogenous echogenic mass, and so can distinguish it from a mesenteric cyst. Computed Tomography (CT) is the standard imaging of diagnosis and shows homogenous tumor of adipose tissue. The treatment is surgery and the prognosis is better. CONCLUSION: The mesenteric is an uncommon location of lipoma. When there is small bowel obstruction with intra-abdominal mass, the mesenteric lipoma could be recalled.

Small bowel adenocarcinoma a rare cause of upper gastrointestinal obstruction (a case report and literature review).

INTRODUCTION: Small bowel adenocarcinoma is a rare but increasing disease. It poses both a diagnostic and therapeutic challange. Small bowel adenocarcinoma is a rare cause of small bowel obstruction. We present the case of a patient admitted to our emergency department for a bowel obstruction due to a mass of the jejunum and whose anatomopathological diagnosis was adenocarcinoma. PATIENT AND METHOD: It is a 62-year-old woman with unparticular history, admitted to the emergency of visceral surgery of Ibn Rochd University Hospital for subocclusive syndrome evolving for one year, with early postprandial vomiting becoming stenosing two months ago. The abdominal CT scan showed thickening jejunal wall of 46 mm with upstream distension. She underwent a segmental bowel resection of 50 cm of small bowel with 3 cm stenotic mass located at 40 cm from the duodenojejunal angle. The pathophysiology revealed an invasive liberkhunian adenocarcinoma. The postoperative follow-up was simple, feeding allowed at D4 with discharge allowed at D6 and functional improvement at the time of the control performed three months after the intervention. DISCUSSION: Small bowel adenocarcinoma is rare and represents only 1-3% of all gastrointestinal cancers. The incidence of SBA is 24 to 66 times lower than that of colorectal cancer (CRC). Due to its non-specific clinical manifestation and less accessible location, SBA is diagnosed at an advanced stage, and often at specimen analysis. The treatment is resection and the overall survival is increased when diagnostic is early made. CONCLUSION: Small bowel adenocarcinoma is a rare but increasing cause of gastrointestinal malignancy, being both a diagnostic and therapeutic challenge. In front of the occlusive syndrome of small bowel appearance, adenocarcinoma must be ruled out.

Fournier's gangrene: Seven years of experience in the emergencies service of visceral surgery at Ibn Rochd University Hospital Center.

INTRODUCTION: This work aims to describe and discuss the epidemiological, clinical, therapeutic and evolution of Fournier's gangrene. MATERIALS AND METHODS: Case series with retrospective data collection of patients treated for Fournier's gangrene between January 2010 and March 2017. The main etiologies, risk factors, postoperative complications outcomes and long term follow up results were analyzed. RESULTS: Eight four (84) patients were recruited. The average age of our patients was 49 years (with limits of 20-76), the male gender dominates our series (83.33%) with a sex ratio of 5 M/1W, the most frequently found risk factor was diabetes mellitus (37%). The most common etiology was anal abscesses (32%). The average time to consultation was 8 days (limits ranges from 3 to 30 days). All patients were admitted at a necrosis stage (100%). Anemia was identified in 85% of cases. The low platelets were noticed in 44.03% of cases. Hypoalbuminemia was found in 93% of cases. All patients (100%) benefited resuscitation initially and antibiotic therapy on their admission. They received emergency surgical debridement with a cleansing stoma. The average length of hospital stay was 13 days and complications occurred in 33% of cases. The mortality rate was 7.14%. CONCLUSION: Fournier's gangrene is a medico-surgical emergency with a high morbidity and mortality rate. Early diagnosis as well as antibiotic therapy and the quality of debridement save the patients.

Bartholin's gland abscess a rare cause of rectovaginal fistula: A case report and literature review.

INTRODUCTION: Rectovaginal fistula (RVF) is an abnormal communication between the vagina and the rectum. RVFs caused by Bartholin's gland infection are very rare. We present the case of recurrent rectovaginal fistula complicating a bartholin's gland abcess successfully treated with a Martius flap. The aim of this work is to demonstrate the possibility of complication of bartholin's gland infection by a rectovaginal fistula and the efficacy of Martius flap procedure for recurrent RVF of the low third part of the vagina. OBSERVATION: It is a 30-year-old woman admitted to our department for recurrent RVF due to an abscess of the Bartholin's glands. She was previously treated with a vaginal advancement flap which failed with persistence of the fistula and its symptoms. The patient underwent a RVF repair by Martius flap with complete healing of the fistula. DISCUSSION: Rectovaginal fistula is a complex pathology with psycho-social, individual, family, religious and ethno-environmental repercussions. Its main aetiologies are obstetric, rectal surgery. Several techniques including the vaginal or anal advancement flap and the Martius flap are used for the treatment of rectovaginal fistulas. For recurrent fistulas, the Martius flap seems to be the most indicated with better results. CONCLUSION: Rectovaginal fistula remain a challenge for surgeons and have major psycho-socio-economic repercussions for the patient. The complication of Bartholin's gland infection by rectovaginal fistula is rare. The Martius flap technique is the method of choice for recurrent rectovaginal fistulas of the lower third of the vagina or in association with other pathologies.

Correlation of Three Dimensions of Palate with Maxillary Arch Form and Perimeter as Predictive Measures for Orthodontic and Orthognathic Surgery.

Hard palate is regarded as an important part of the human skull, which contributes to the separation of the oral and nasal cavities. The aims of the study were to investigate the morphology of the hard palate in order to create a general guideline of three-dimensional values of the palate in a Kurdish sample in the city of Sulaimani as well as determining the possible correlations between different palatal parameters in class I malocclusion with the maxillary arch form and perimeter. A retrospective study design was adopted by collecting 100 study models of orthodontic patients aged 16-24 years old attending different private dental clinics in the city of Sulaimani seeking orthodontic management. In this study, three-dimensional palatal measurements including depth, length, and width were measured in an attempt to discover their correlation with each maxillary arch form and perimeter. Additionally, measurements of inter-molar width, inter-canine width, and arch perimeter were carried out. About two-thirds of those seeking orthodontic treatment were females. Nearly 80% of the study sample had narrow palate followed by 15 and 5% of intermediate palate and broad palate, respectively. In regard to arch form, almost 90% of subjects were with tapered maxillary arch form and 10% of them with oval arch form. Males had increased dimensions compared to females, with significant differences, except in palatal depth in the molar area, and palatine height index, in which females showed increased dimensions than males but the differences were statistically non-significant. A strong positive correlation was observed between arch form and canine depth. In regard to arch perimeter, a strong negative correlation was found with molar depth and a medium positive correlation with each of canine depth, palatal width, and palatal length.

Post-traumatic diaphragmatic rupture with pericardial denudation: A case report.

INTRODUCTION: Post-traumatic diaphragmatic rupture is a lesion of variable severity. It is a rare and difficult to diagnose pathology, it has been found in 0.4% of all traumatized patients and in 1.9% of blunt traumas. It can be associated with abdominal andthoracic lesions, particularly cardiac, which can be life-threatening. MATERIALS AND METHODS: Our work is a retrospective case report with a descriptive aim concerning a patient operated for a post-traumatic diaphragmatic rupture within the department of general surgery of CHU Ibn Rochd Casablanca. This work has been reported in line with the SCARE 2020 criteria (17). CASE PRESENTATION: A 60-year-old patient was admitted to the visceral surgical emergency department following a work accident (crushing between two carts) causing a thoraco-abdominal impact point trauma without initial loss of consciousness, nor externalized digestive hemorrhage or associated signs, but with a general condition alteration. The patient was conscious, dyspneic with a blood pressure of 100/50 mmHg and afebrile. Physical examination showed diffuse abdominal sensibility. The thoraco-abdomino-pelvic CT scan revealed the presence of a left thoracic hernia with gastric, colic and epiploic contents through a lateral defect of the left diaphragmatic dome. The decision was to directly send the patient to the operating room. Exploration found a large left diaphragmatic breach of 20 cm, a denudation of the pericardia, a medium-abundant hemoperitoneum and a hematoma of the right mesocolon. The procedure consisted of right hemicolectomy with ileocolic anastomosis, treatment of a diaphragmatic breach with a 2-silk raphia, thoracic drainage with a Joly drain, pericardial drainage with a Joly drain, pre-anastomotic drainage with 2 delbet slides, drainage of the Douglas and left subthreshold with 2 Salem catheters. The post-operative follow-up was simple. DISCUSSION: Diaphragmatic rupture is a rare and difficult to diagnose condition. Traumatic diaphragmatic rupture (TDR) was found in 0.4% of all traumatized patients and in 1.9% of blunt trauma. Associated lesions of the spleen, liver and/or lungs were found in more than 30% of cases, with an overall mortality rate of 26.8% (1). Pericardial rupture following blunt chest trauma is rare and associated with a high mortality rate ranging from 30% to 64% (9). The physiopathology of this type of injury is not well understood, but the most accepted hypothesis describes an increase in intra-abdominal pressure due to a blunt creating a sufficiently high-pressure gradient between the chest and the abdomen to cause a diaphragmatic rupture. The common clinical symptoms of a diaphragmatic rupture are a marked respiratory distress and diffuse abdominal pain but it can be asymptomatic. Medical imaging exams visualize the ascended organs but it's more difficult to visualize the rupture itself. The chest X-ray is currently the first examination to be requested (4) and also helps in the diagnosis of injuries and diaphragm rupture (13). Surgical treatment includes the reduction of any visceral hernia, repair of the diaphragm and restoration of circulation, breathing and digestive functions. Laparotomy is generally used because of the complete exploration of the abdominal viscera, although it is easier to reduce herniated tissue and repair the diaphragm. CONCLUSION: Diaphragmatic rupture with denudation of the heart is rare with poor prognosis and requires emergency surgery with close postoperative monitoring in the intensive care setting. SUMMARY: Post-traumatic diaphragmatic rupture is a lesion of variable severity. It is a rare and difficult to diagnose pathology, it has been found in 0.4% of all traumatized patients and in 1.9% of blunt traumas. The lesions are more frequent in the left diaphragmatic dome compared to the right one, and exceptionally bilateral. Pericardial rupture following blunt chest trauma is rare and associated to a high mortality rate. It is often unrecognized and goes unnoticed in the acute phase. The most common clinical symptoms of diaphragmatic ruptures are respiratory distress and diffuse abdominal pain, as it can be asymptomatic. Its diagnosis is essentially radiological using CT scan, and requires emergency surgical treatment as soon as the diagnosis is suspected, in order to avoid the dreaded complications. Traumatic diaphragmatic rupture remains a diagnostic and therapeutic challenge. We report the case of a patient who presented a post-traumatic diaphragmatic rupture with pericardial damage operated in the visceral emergency department at the Ibn Rochd Hospital c in Casablanca, Morocco.

Lactococcus lactis ssp lactis a rare cause of liver abscesses: A case report and literature review.

INTRODUCTION: We present a liver abscess due to Lactococcus lactis ssp lactis. CASE PRESENTATION: It is a 27-year-old male patient without history who presented the right hypochondrium pain over 10 days. The physical examination noted right hypochondrium pain and hépatomegally. The ultrasound showed hepatomegaly with liver abcess for the segments IV and V as well as VII and VIII measuring 13 × 8 cm and 7.6 × 4.3 cm respectively. A computed tomography (CT) revealed an abscess for segments IV and V and VI and VII measuring respectively 107 × 89 mm and 55 × 50 mm. He underwent a surgical drainage after a radiologic drainage and antibiotherapy failure with success. DISCUSSION: Liver abscesses are rare; affect men over 60 years with co-morbidities and those due to L. Lactis ssp lactis are exceptional. Their prevalence is 0.29-1.47% in series of autopsies and 0.008 to 0.16% in hospitalized patients. The most frequently found germs are gram-negative bacilli (40-60%) and anaerobic bacteria (40-50%). Ultrasound and CT scan make the diagnosis in 90% of cases and orients to the etiology. Percutaneous drainage is the first line for treatment, surgical drainage is reserved for percutaneous drainage failures. CONCLUSION: Liver abscess due to Lactococcus lactis ssp lactis is very rare. The clinic, diagnostic methods and treatment of this abscess are identical to other abscesses due to other etiologies. The antibiotics and percutaneous drainage of abscesses have improved the death rate from 40% to 10%-25%.

Acute pancreatitis associated with Cushing syndrome - A case report and literature review.

INTRODUCTION: Cushing's syndrome (CS) is a rare and severe disease. Acute pancreatitis is the leading cause of hospitalization. The association of the two disease is rare and uncommon. We report the case of a 37-year-old woman admitted in our service for acute pancreatitis and whose Cushing syndrome was diagnosed during hospiatilisation. The aim of this work is to try to understand the influence of de Cushing in acute pancreatitis and to establish a causative relationship between the two diseases. OBSERVATION: It is a 37-year-old woman with a history of corticosteroid intake for six months, stopped three months ago who consulted for epigastralgia and vomiting. The physical exam found epigastric sensitivity with Cushing syndrome symptoms. A CT scan revealed acute edematous-interstitial pancreatitis stage E of Balthazar classification. 24 h free cortisol of 95 µg/24 h and cortisolemia of 3.4 µg/dl. The patient was treated symptomatically and referred after to endocrinology service for further treatment. CONCLUSION: The association with acute pancreatitis and CS is rare and uncommon. Although detailed studies and evidence are lacking, it can therefore be inferred that CS is one of the risk factors for the onset of acute pancreatitis. The medical treatment and management of acute pancreatitis in those patients do not differ from other pancreatitis of any etiologies.

Isolating Pathogen-Specific Human Monoclonal Antibodies (hmAbs) Using Bacterial Whole Cells as Molecular Probes.

The immunoglobulin capture assay (ICA) enables the enrichment for pathogen-specific plasmablasts from individuals with a confirmed adaptive immune response to vaccination or disseminated infection. Only single recombinant antigens have been used previously as probes in this ICA and it was unclear whether the method was applicable to complex probes such as whole bacterial cells. Here, we describe the enrichment of plasmablasts specific for polysaccharide and protein antigens of both Streptococcus pneumoniae and Neisseria meningitidis using whole formalin-fixed bacterial cells as probes. The modified ICA protocol described here allowed for a pathogen-specific hmAb cloning efficiency of >80%.

Bacterial Vaccine Antigen Discovery in the Reverse Vaccinology 2.0 Era: Progress and Challenges.

The ongoing, and very serious, threat from antimicrobial resistance necessitates the development and use of preventative measures, predominantly vaccination. Polysaccharide-based vaccines have provided a degree of success in limiting morbidity from disseminated bacterial infections, including those caused by the major human obligate pathogens, Neisseria meningitidis, and Streptococcus pneumoniae. Limitations of these polysaccharide vaccines, such as partial coverage and induced escape leading to persistence of disease, provide a compelling argument for the development of protein vaccines. In this review, we briefly chronicle approaches that have yielded licensed vaccines before highlighting reverse vaccinology 2.0 and its potential application in the discovery of novel bacterial protein vaccine candidates. Technical challenges and research gaps are also discussed.

Cross-Reactive Bactericidal Antimeningococcal Antibodies Can Be Isolated From Convalescing Invasive Meningococcal Disease Patients Using Reverse Vaccinology 2.0.

The threat from invasive meningococcal disease (IMD) remains a serious source of concern despite the licensure and availability of vaccines. A limitation of current serogroup B vaccines is the breadth of coverage afforded, resulting from the capacity for extensive variation of the meningococcus and its huge potential for the generation of further diversity. Thus, the continuous search for candidate antigens that will compose supplementary or replacement vaccines is mandated. Here, we describe successful efforts to utilize the reverse vaccinology 2.0 approach to identify novel functional meningococcal antigens. In this study, eight broadly cross-reactive sequence-specific antimeningococcal human monoclonal antibodies (hmAbs) were cloned from 4 ml of blood taken from a 7-month-old sufferer of IMD. Three of these hmAbs possessed human complement-dependent bactericidal activity against meningococcal serogroup B strains of disparate PorA and 4CMenB antigen sequence types, strongly suggesting that the target(s) of these bactericidal hmAbs are not PorA (the immunodominant meningococcal antigen), factor-H binding protein, or other components of current meningococcal vaccines. Reactivity of the bactericidal hmAbs was confirmed to a single ca. 35 kDa protein in western blots. Unequivocal identification of this antigen is currently ongoing. Collectively, our results provide proof-of-principle for the use of reverse vaccinology 2.0 as a powerful tool in the search for alternative meningococcal vaccine candidate antigens.

Glomerulonephritis disease pattern at Saudi tertiary care center.

OBJECTIVE: To assess changes in the pattern of glomerular diseases to help guide optimal allocation of resources, to focus future reasearch, and improve outcomes. Methods: A retrospective chart review was conducted on kidney biopsies taken between 2007 and 2016 at a single tertiary care center in Saudi Arabia (King Fahad Medical City, Riyadh) to evaluate the prevalence and pattern of glomerulonephritis (GN). Results: The most common primary GN in 102 biopsies from adult patients with a mean age of 28.9 ± 13.6 years and 40.2% female, was focal and segmental glomerulosclerosis (35.3%). Among 64 patients with systemic lupus erythematosus associated nephritis, of whom most (82.8%) were female, lupus nephritis (LN) 4 (46.9%), and (LN) 3 (32.8%) were the most common lupus nephritis classes. Conclusion: Establishing prospective GN registries from which robust diagnosis, treatment, and outcomes data can be acquired is warranted; however, registry development and maintenance are often precluded by resource limitations. Accordingly, retrospective analysis of administrative data will continue to provide important complementary information on GN epidemiology.

Association of single nucleotide polymorphisms in pro-inflammatory cytokine and toll-like receptor genes with pediatric hematogenous osteomyelitis.

Hematogenous osteomyelitis (HO) is a bone infection wherein bacteria penetrate to the bone through the blood stream. Several single nucleotide polymorphisms (SNPs) have been associated with susceptibility to infectious diseases. In this study, we investigated the contribution of SNPs in interleukin (IL)-1B1 (rs16944), IL1A (rs1800587), IL1B (rs1143634), toll-like receptor (TLR)-2 (rs3804099), TLR4 (rs4986790), TLR4 (rs4986791), IL1R (rs2234650), tumor necrosis factor (TNF)-α (rs1800629), TNF (rs361525), and IL1RN (rs315952) towards the development of HO in Saudi patients and compared to healthy controls. Fifty-two patients diagnosed with HO and 103 healthy individuals were genotyped. The frequencies of genotypes GG (rs16944) and AA (rs16944) were lower and higher in patients [odds ratio (OR) = 0.34, Pc = 0.05] and controls (OR = 1.33, Pc = 0.05), respectively, suggesting that SNPs at this locus could alter HO susceptibility. In addition, the patients and controls exhibited lower and higher frequencies of the alleles G (rs16944) (OR = 0.43, Pc = 0.007) and A (rs16944) (OR = 2.32, Pc = 0.007), respectively. The expression of alleles C (rs3804099) and T (rs3804099) were higher in patients (OR = 2.05, Pc = 0.04) and controls (OR = 0.49, Pc = 0.04), respectively. In conclusion, SNPs at rs16944 and rs3804099 were found to be associated with HO in the Saudi population.

Investigation of polymorphisms in anti-inflammatory cytokine genes in hematogenous osteomyelitis.

Osteomyelitis is a progressive bone infection disease caused by destructive immunological inflammatory reactions following new bone formation. Anti-inflammatory cytokines are a series of immunoregulatory molecules that control the pro-inflammatory cytokine response. In this study, we investigated 9 single nucleotide polymorphisms in 5 different cytokine/cytokine receptor genes in hematogenous osteomyelitis (HO) patients, and compared their outcomes with normal healthy individuals. Sequence-specific forward and reverse primers and two TaqMan® MGB probes with dyes (VIC™ and FAM™) that specifically detect Allele 1 and Allele 2 of each SNP were utilized. The genotypes CC (P = 0.009) and CT (P = 0.041) of SNP rs2070874, and alleles A (P = 0.044) and G of SNP rs1800871 were significantly different between the patients and healthy controls. The expression of the CC genotype or C allele at rs2070874 was a risk factor for HO development, with higher frequencies of CT and T being found in the control samples. The expression of the A allele of rs1800871 was also significantly higher in patients than in controls, and was therefore considered a risk factor.

Investigation into the Antigenic Properties and Contributions to Growth in Blood of the Meningococcal Haemoglobin Receptors, HpuAB and HmbR.

Acquisition of iron from host complexes is mediated by four surface-located receptors of Neisseria meningitidis. The HmbR protein and heterodimeric HpuAB complex bind to haemoglobin whilst TbpBA and LbpBA bind iron-loaded transferrin and lactoferrin complexes, respectively. The haemoglobin receptors are unevenly distributed; disease-causing meningococcal isolates encode HmbR or both receptors while strains with only HpuAB are rarely-associated with disease. Both these receptors are subject to phase variation and 70-90% of disease isolates have one or both of these receptors in an ON expression state. The surface-expression, ubiquity and association with disease indicate that these receptors could be potential virulence factors and vaccine targets. To test for a requirement during disease, an hmbR deletion mutant was constructed in a strain (MC58) lacking HpuAB and in both a wild-type and TbpBA deletion background. The hmbR mutant exhibited an identical growth pattern to wild-type in whole blood from healthy human donors whereas growth of the tbpBA mutant was impaired. These results suggest that transferrin is the major source of iron for N. meningitidis during replication in healthy human blood. To examine immune responses, polyclonal antisera were raised against His-tagged purified-recombinant variants of HmbR, HpuA and HpuB in mice using monolipopolysaccharide as an adjuvant. Additionally, monoclonal antibodies were raised against outer membrane loops of HmbR presented on the surface of EspA, an E. coli fimbrial protein. All antisera exhibited specific reactivity in Western blots but HmbR and HpuA polyclonal sera were reactive against intact meningococcal cells. None of the sera exhibited bactericidal activity against iron-induced wild-type meningococci. These findings suggest that the HmbR protein is not required during the early stages of disease and that immune responses against these receptors may not be protective.

Genomic and global approaches to unravelling how hypermutable sequences influence bacterial pathogenesis.

Rapid adaptation to fluctuations in the host milieu contributes to the host persistence and virulence of bacterial pathogens. Adaptation is frequently mediated by hypermutable sequences in bacterial pathogens. Early bacterial genomic studies identified the multiplicity and virulence-associated functions of these hypermutable sequences. Thus, simple sequence repeat tracts (SSRs) and site-specific recombination were found to control capsular type, lipopolysaccharide structure, pilin diversity and the expression of outer membrane proteins. We review how the population diversity inherent in the SSR-mediated mechanism of localised hypermutation is being unlocked by the investigation of whole genome sequences of disease isolates, analysis of clinical samples and use of model systems. A contrast is presented between the problematical nature of analysing simple sequence repeats in next generation sequencing data and in simpler, pragmatic PCR-based approaches. Specific examples are presented of the potential relevance of this localized hypermutation to meningococcal pathogenesis. This leads us to speculate on the future prospects for unravelling how hypermutable mechanisms may contribute to the transmission, spread and persistence of bacterial pathogens.

Phase variation mediates reductions in expression of surface proteins during persistent meningococcal carriage.

Asymptomatic and persistent colonization of the upper respiratory tract by Neisseria meningitidis occurs despite elicitation of adaptive immune responses against surface antigens. A putative mechanism for facilitating host persistence of this bacterial commensal and pathogen is alterations in expression of surface antigens by simple sequence repeat (SSR)-mediated phase variation. We investigated how often phase variation occurs during persistent carriage by analyzing the SSRs of eight loci in multiple isolates from 21 carriers representative of 1 to 6 months carriage. Alterations in repeat number were detected by a GeneScan analysis and occurred at 0.06 mutations/gene/month of carriage. The expression states were determined by Western blotting and two genes, fetA and nadA, exhibited trends toward low expression states. A critical finding from our unique examination of combinatorial expression states, "phasotypes," was for significant reductions in expression of multiple phase-variable surface proteins during persistent carriage of some strains. The immune responses in these carriers were examined by measuring variant-specific PorA IgG antibodies, capsular group Y IgG antibodies and serum bactericidal activity in concomitant serum samples. Persistent carriage was associated with high levels of specific IgG antibodies and serum bactericidal activity while recent strain acquisition correlated with a significant induction of antibodies. We conclude that phase-variable genes are driven into lower expression states during long-term persistent meningococcal carriage, in part due to continuous exposure to antibody-mediated selection, suggesting localized hypermutation has evolved to facilitate host persistence.

Prevalence and phase variable expression status of two autotransporters, NalP and MspA, in carriage and disease isolates of Neisseria meningitidis.

Neisseria meningitidis is a human nasopharyngeal commensal capable of causing life-threatening septicemia and meningitis. Many meningococcal surface structures, including the autotransporter proteins NalP and MspA, are subject to phase variation (PV) due to the presence of homopolymeric tracts within their coding sequences. The functions of MspA are unknown. NalP proteolytically cleaves several surface-located virulence factors including the 4CMenB antigen NhbA. Therefore, NalP is a phase-variable regulator of the meningococcal outer membrane and secretome whose expression may reduce isolate susceptibility to 4CMenB-induced immune responses. To improve our understanding of the contributions of MspA and NalP to meningococcal-host interactions, their distribution and phase-variable expression status was studied in epidemiologically relevant samples, including 127 carriage and 514 invasive isolates representative of multiple clonal complexes and serogroups. Prevalence estimates of >98% and >88% were obtained for mspA and nalP, respectively, with no significant differences in their frequencies in disease versus carriage isolates. 16% of serogroup B (MenB) invasive isolates, predominately from clonal complexes ST-269 and ST-461, lacked nalP. Deletion of nalP often resulted from recombination events between flanking repetitive elements. PolyC tract lengths ranged from 6-15 bp in nalP and 6-14 bp in mspA. In an examination of PV status, 58.8% of carriage, and 40.1% of invasive nalP-positive MenB isolates were nalP phase ON. The frequency of this phenotype was not significantly different in serogroup Y (MenY) carriage strains, but was significantly higher in invasive MenY strains (86.3%; p<0.0001). Approximately 90% of MenB carriage and invasive isolates were mspA phase ON; significantly more than MenY carriage (32.7%) or invasive (13.7%) isolates. This differential expression resulted from different mode mspA tract lengths between the serogroups. Our data indicates a differential requirement for NalP and MspA expression in MenB and MenY strains and is a step towards understanding the contributions of phase-variable loci to meningococcal biology.