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The article presents a hypothesis on the autoimmunity elicited by the chemokine response to adenovirus vector vaccines may underlie vaccine-induced immune thrombotic thrombocytopaenia. Topics include vaccination has formed the critical component of the public health response for the prevention of severe illness and death with multiple different vaccines; and increase in unusual thrombotic events temporally associated with the AstraZeneca vaccine.

The article focuses on neuritin has unmasked as a checkpoint for the pathogenesis of allergy and autoimmunity. Topics include the follicular regulatory T cells (Tfr) in the germinal centre suppress the development of B-cell-driven autoimmunity and IgE-mediated allergic sensitisation via their secretion of the neuropeptide neuritin, and the unmasks a lack of neuritin as a previously unrecognised critical contributor to the development of allergic and autoimmune disease.

The article presents a case study of a 45-year-old man with drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome who was diagnosed with symptoms like maculopapular exanthema. Topics include dapsone-induced hypersensitivity syndrome (DHS) and the association of HLA-B 13:01 antigens with DHS.

Host hepatitis C virus (HCV)-specific T cell responses and the ability of the virus to escape this response are important correlates of infection outcome. Understanding this host-viral interplay has been difficult given the often asymptomatic nature of acute HCV infection. We studied a recent transmission case to determine whether adapted viral strains can be transmitted and influence the recipient's anti-HCV T cell response. The diversity of viral populations was examined using next-generation sequencing, and HCV-specific T cell interferon (IFN)--γ responses were assessed using a peptide panel representing the autologous viruses. HCV-specific T cell responses in the source were directed against peptides that did not match the dominant autologous virus but rather low-frequency variants, implying existing viral adaptation in the source strain. Most HCV T cell epitopes that elicited an IFN-γ response in the source did not in the recipient, despite the pair sharing human leukocyte antigen alleles that govern antigen presentation and similar autologous viruses. Intrahost HCV variation in the recipient fell within predicted T cell epitopes, suggesting alternative targets of the immune response. These data suggest that transmission of adapted viral species can direct the host's HCV-specific immune response profile during acute infection. [ABSTRACT FROM AUTHOR]

Background: Fifty-five percent of individuals with HLA-B*57:01 exposed to the antiretroviral drug abacavir develop a hypersensitivity reaction (HSR) that has been attributed to naïve T-cell responses to neo-antigen generated by the drug. Immunologically confirmed abacavir HSR can manifest clinically in less than 48 hours following first exposure suggesting that, at least in some cases, abacavir HSR is due to re-stimulation of a pre-existing memory T-cell population rather than priming of a high frequency naïve T-cell population. Methods: To determine whether a pre-existing abacavir reactive memory T-cell population contributes to early abacavir HSR symptoms, we studied the abacavir specific naïve or memory T-cell response using HLA-B*57:01 positive HSR patients or healthy controls using ELISpot assay, intra-cellular cytokine staining and tetramer labelling. Results: Abacavir reactive CD8+ T-cell responses were detected in vitro in one hundred percent of abacavir unexposed HLA-B*57:01 positive healthy donors. Abacavir-specific CD8+ T cells from such donors can be expanded from sorted memory, and sorted naïve, CD8+ T cells without need for autologous CD4+ T cells. Conclusions: We propose that these pre-existing abacavir-reactive memory CD8+ T-cell responses must have been primed by earlier exposure to another foreign antigen and that these T cells cross-react with an abacavir-HLA-B*57:01-endogenous peptide ligand complex, in keeping with the model of heterologous immunity proposed in transplant rejection. [ABSTRACT FROM AUTHOR]

Environmental changes affecting the relationship between the developing immune system and microbial exposure have been implicated in the epidemic rise of allergic disease in developed countries. While early developmental differences in T cell function are well-recognised, there is now emerging evidence that this is related to developmental differences in innate immune function. In this study we sought to examine if differences associated with innate immunity contribute to the altered immune programming recognised in allergic children. Here, we describe for the first time, the association of carriage of the T allele of the tagging single nucleotide polymorphism rs12979860 3 kb upstream of IL28B, encoding the potent innate immune modulator type III interferon lambda (IFN-λ3), and allergy in children (p = 0.004; OR 4.56). Strikingly, the association between rs12979860 genotype and allergic disease is enhanced in girls. Furthermore, carriage of the T allele at rs12979860 correlates with differences in the pro-inflammatory profile during the first five years of life suggesting this contributes to the key differences in subsequent innate immune development in children who develop allergic disease. In the context of rising rates of disease, these immunologic differences already present at birth imply very early interaction between genetic predisposition and prenatal environmental influences. [ABSTRACT FROM AUTHOR]

Background Diagnosis of latent tuberculosis infection (LTBI) is a cornerstone of the health assessment of resettled high incidence populations, particularly in children. Two blood-based interferon g release assays (IGRAs), T-SPOT.TB and QFT-Gold in-tube (QFT-GIT), have greater sensitivity and specificity than the tuberculin skin test (TST), but their performance as screening tools for LTBI in children, especially refugee children, remains unclear. Methods 524 African and ethnic Burmese children, including 107 under 3 years of age, were prospectively enrolled in a comparison of the T-SPOT.TB and QFT-GIT. The TST was also performed in 342 of the children. Results The T-SPOT.TB and QFT-GIT had similar rates of positivity (8% and 10%, respectively) and showed good concordance when both tests gave definitive results (k=0.78; p<0.0001). However, the IGRAs had significant failure rates: 15% of QFT-GIT gave indeterminate results due to failed mitogen response and 14% of T-SPOT.TB results were inconclusive, largely because of insufficient mononuclear leucocyte yields. Failure of the QFT-GIT mitogen response was associated with African ethnicity and co-morbid infections, particularly with helminths. The TST results showed poor concordance (∼50%) with both IGRAs. Conclusions It is reasonable to screen using either IGRA with follow-up by the alternative if the test fails. In general, the QFT-GIT is the preferred option for non-African populations but the T-SPOT.TB is recommended when there are epidemiological and/or clinical high risk factors for TB infection. However, both IGRAs have methodological and performance characteristics that limit their usefulness in refugee children, highlighting the need for continued development of screening strategies. [ABSTRACT FROM AUTHOR]

Multispecific CD8+ T-cell responses are thought to be important for the control of acute hepatitis C virus (HCV) infection, but to date little information is actually available on the breadth of responses at early time points. Additionally, the influence of early therapy on these responses and their relationships to outcome are controversial. To investigate this issue, we performed comprehensive analysis of the breadth and frequencies of virus-specific CD8+ T-cell responses on the single epitope level in eight acutely infected individuals who were all started on early therapy. During the acute phase, responses against up to five peptides were identified. During therapy, CD8+ T-cell responses decreased rather than increased as virus was controlled, and no new specificities emerged. A sustained virological response following completion of treatment was independent of CD8+ T-cell responses, as well as CD4+ T-cell responses. Rapid recrudescence also occurred despite broad CD8+ T-cell responses. Importantly, in vivo suppression of CD3+ T cells using OKT3 in one subject did not result in recurrence of viremia. These data suggest that broad CD8+ T-cell responses alone may be insufficient to contain HCV replication, and also that early therapy is effective independent of such responses. [ABSTRACT FROM AUTHOR]

CMV-specific memory CD8+ T cells accumulate over time to reach high frequencies amongst peripheral blood lymphocytes– a phenomenon termed‘memory inflation’. Using tetramer staining on samples from a large number of subjects and multivariate regression analysis, we were able to relate this to the phenotype of CD8+ T cells. We made the following observations: (i) CD85j (ILT-2/LIR-1) was highly expressed alongside CD57– an established effector memory marker– on CMV-specific CD8+ T cells; (ii) on CD8+ T cells as a whole, with increasing age, CD57 and CD85j (ILT-2/LIR-1) expression increased whereas CCR7 expression decreased, indicating increasing maturation of the total CD8+ T-cell compartment with age; (iii) unit increases in the percentage of CMV-specific CD8+ T cells expressing CD57 and CD85j (ILT-2/LIR-1) were associated with incremental expansion of these T-cell populations; (iv) CMV seropositivity is associated with a marked effect on the overall phenotype of CD8+ T cells (at any given age, CMV seropositivity is associated with an 18.7% increase in CD85j (ILT-2/LIR-1) expression); and (v) from our observations we estimated from this an apparent‘ageing effect’ of CMV on CD8+ T cells of 35.4 years. The data presented are consistent with a predictable, unidirectional and linear model of virus specific T-cell differentiation and maturation. [ABSTRACT FROM AUTHOR]

Recent advances in class II tetramer staining technology have allowed reliable direct ex vivo visualization of antigen-specific CD4 T cells. In order to define the frequency and phenotype of a prototype response to a nonporsistent pathogen, we have used such techniques to analyze influenza virus-specific memory CD4 T cells directly from blood. These responses are stably detectable ex vivo at low frequencies (range, 0.00012 to 0.0061% of CD4 T cells) and display a distinct ‘central memory’ CD62L+ phenotype. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) has infected over 170 million people world wide, and in the majority sets up a chronic infection associated with hepatic inflammation. How it evades host immunity, particularly CD8+ T cells (CTL) is unclear, but two major factors are likely to operate, viral escape mutation and T cell exhaustion. We have investigated the role of CTL in control of infection during acute disease using Class I peptide tetramers. Although the immune response is quite diverse and numerous epitopes can be targeted, we observe that, especially during acute disease, one epitope (NS3 1073–81) is commonly recognised in HLA-A2 positive individuals. However, the levels of response to this epitope (and others) are very much lower if persistence is established. We examined in detail whether the cause of this low level of reactivity is due to mutation within the epitope. We find that, in fact this epitope is highly conserved during chronic infection, at a clonal level, between individuals, and over time. Thus, although variation within the epitope does occur, lack of reactivity in peripheral blood against this epitope in chronic disease, and loss of control of virus cannot be explained entirely by viral escape. Escape through mutation probably does play an important role in persistence of HCV, but we also discuss other mechanisms which lead to attenuation of T cell responses which may be important in determining the outcome. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) readily causes a persistent infection, although some individuals spontaneously control infection. ‘Successful’ immune responses appear to be multi-specific and sustained-including a major role for CD4+ T cells. Some antiviral CD8+ T cells show reduced capacity to secrete antiviral cytokines either temporarily (‘stunning’) or in the long term (‘stunting’). The co-ordination of multiple immune effector functions may be required to gain control of HCV. [Copyright &y& Elsevier]

Viruses have ‘studied’ immunology over millions of years of coevolution with their hosts. During this ongoing education they have developed countless mechanisms to escape from the host's immune system.To illustrate the most common strategies of viral immune escape we have focused on two murine models of persistent infection, lymphocytic choriomeningitis virus (LCMV) and murine cytomegalovirus (MCMV).LCMV is a fast replicating small RNA virus with a genome prone to mutations. Therefore, LCMV escapes from the immune system mainly by two strategies: ‘speed’ and ‘shape change’. At the opposite extreme, MCMV is a large, complex DNA virus with a more rigid genome and thus the strategies used by LCMV are no option. However, MCMV has the coding capacity for additional genes which interfere specifically with the immune response of the host. These escape strategies have been described as ‘camouflage’ and ‘sabotage’. Using these simple concepts we describe the spectrum of viral escapology, giving credit not only to the researchers who uncovered this fascinating area of immunology but also to the viruses themselves, who still have a few lessons to teach. [ABSTRACT FROM AUTHOR]

Aim: Penicillin allergy accounts for the majority of all reported adverse drug reactions in adults and children. Foregoing first‐line antibiotic therapy due to penicillin allergy label is associated with an increased prevalence of infections by resistant organisms and longer hospitalisation. Clinician awareness of allergy assessment, referral indications, management of allergy and anaphylaxis is therefore vital but globally lacking. We aim to assess the knowledge of penicillin allergy, assessment and management in Western Australian health professionals. Methods: An anonymous survey was distributed to pharmacists, nurses and physicians within Western Australian paediatric and adult Hospitals, Community and General Practice. Results: In total, 487/611 were completed and included in the statistical analysis. Only 62% (301/487) of respondents routinely assessed for patient medication allergies. Of those who assessed allergy, 9% (28/301) of respondents met the Australian standards for allergy assessment. Only 22% (106/487) of participants correctly cited all indications for management with adrenaline in anaphylaxis to antibiotics and 67% (197/292) of physicians rarely or never referred to an allergy service. Paediatric clinicians had an increased understanding of allergy assessment and anaphylaxis management. Recent penicillin allergy education within a 5‐year period led to significant improvements in allergy knowledge. Conclusion: Overall, knowledge, assessment and management of penicillin allergies among practitioners in Western Australia are currently inadequate in adults and paediatric clinicians to provide safe and effective clinical care. The implementation of a targeted education program for WA health professionals is urgently required and is expected to improve clinician knowledge and aid standardised penicillin assessment (de‐labelling) practices. [ABSTRACT FROM AUTHOR]

Drug reaction with eosinophilia and systemic symptoms (DRESS) syndrome, also known as drug induced hypersensitivity (DiHS) syndrome is a severe delayed hypersensitivity reaction with potentially fatal consequences. Whilst recognised as T cell-mediated, our understanding of the immunopathogenesis of this syndrome remains incomplete. Here, we discuss models of DRESS, including the role of human leukocyte antigen (HLA) and how observations derived from new molecular techniques adopted in key studies have informed our mechanism-based understanding of the central role of Herpesviridae reactivation and heterologous immunity in these disorders. [ABSTRACT FROM AUTHOR]

In our patient's case, her rash began 7 days after commencement of the suspected culprit drug, phenobarbitone which is consistent with early-onset DRESS syndrome.4 Drugs most implicated are antiepileptics (50%), including phenobarbitone (11.5%). GLO:81V/01nov22:jpc16078-fig-0002.jpg PHOTO (COLOR): 2 Patient's drug hypersensitivity timeline. gl She was advised to avoid phenobarbitone and vancomycin lifelong and to avoid amoxicillin, amoxicillin/clavulanic acid, trimethoprim and sulfonamides until further drug patch testing. Early accurate diagnosis of drug reaction with eosinophilia and systemic symptoms (DRESS) can be challenging in patients with a rash on multiple new medications with an associated viral infection. [Extracted from the article]

The lung achieves an efficient gas exchange between a complex non‐sterile atmosphere and the body via a delicate and extensive epithelial surface, with high efficiency because of elastic deformation allowing for an increase and decrease in volume during the process of breathing and because of an extensive vasculature which aids rapid gas diffusion. The lungs' large surface area exposes the organ to a continual risk of damage from pathogens, toxins or irritants; however, lung damage can be rapidly healed via regenerative processes that restore its structure and function. In response to sustained and extensive damage, the lung is healed via a non‐regenerative process resulting in scar tissue which locally stiffens its structure, which over time leads to a serious loss of lung function and to increasing morbidities. This review discusses what is known about the factors which influence whether a lung is healed by regeneration or repair and what potential new therapeutic approaches may positively influence lung healing. [ABSTRACT FROM AUTHOR]

We investigated whether a unique immune response was instigated with the development of oral tongue squamous cell carcinomas (OTSCC), with/without nodal involvement, with/without recurrent metastatic disease, or within tumor involved nodes. One hundred and ten formalin‐fixed paraffin‐embedded samples were collected from a retrospective cohort of 67 OTSCC patients and 10 non‐cancerous tongue samples. Targets including CD4, CD8, FOXP3, PD‐L1, and PD‐1 were analyzed by immunohistochemistry. The Nanostring PanCancer Immune Profiling Panel was used for gene expression profiling. Data were externally validated in the The Cancer Genome Atlas (TCGA) head and neck (HNSCC), melanoma and lung squamous cell carcinoma (LSCC) cohorts. A 24‐immune gene signature was identified that discriminated more aggressive OTSCC cases, and although not prognostic in HNSCC was associated with survival in other TCGA cohorts (improved survival for melanoma, P <.001 and worse survival for LSCC, P =.038). OTSCC exhibited concordant gene and immunohistochemical (IHC) features characterized by a TH‐2 biased, proinflammatory profile with upregulated B cell and neutrophil gene activity and increased CD4, FOXP3, and PD‐L1 expression (P <.001 for all by IHC). Compared to less advanced disease, nodal involvement and recurrent OTSCC did not induce a different immune response although recurrent disease was characterized by significantly higher PD‐L1 expression (P =.004 by SP263, P =.013 by 22C3, P =.004 for gene expression). Identification of a gene signature associated with different prognostic effects in other cancers highlights common pathways of immune dysregulation that are impacted by the tumor origin. The significant immunosuppressive signaling in OTSCC indicates primary failure of immune system to control carcinogenesis emphasizing the need for early, combination therapeutic approaches. [ABSTRACT FROM AUTHOR]

Childhood allergy is common, and increasing. Many children are incorrectly labeled as having allergy or adverse drug reactions. This can pose a dilemma for anesthetists and lead to a change in practice or drug selection. We review the pathophysiology of hypersensitivity reactions and the implications for anesthesia of food allergy, atopy, and family history of allergy in children. The epidemiology of anaphylaxis is discussed. We discuss the common triggers of perioperative anaphylaxis in children and explore emerging triggers including chlorhexidine and sugammadex. Accurate data on pediatric perioperative anaphylaxis is limited worldwide, with marked geographic variation. This highlights the need for accurate local, district and/or nationwide incident reporting. The clinical features, diagnosis, and management of anaphylaxis under anesthesia are discussed. We review the process of expert allergy testing following a suspected case of anaphylaxis to guide future safe anesthesia administration. The preoperative consultation is an opportunity for referral for allergy testing to allow de‐labeling. This has the potential for improved antibiotic stewardship and more effective treatment with first‐line therapeutic agents. [ABSTRACT FROM AUTHOR]

On average, how many individual consultations/visits on average are patients required to attend for entire penicillin allergy delabelling process (including history taking, allergy testing, provocation testing etc.)a. If your centre performs penicillin skin prick/intradermal testing, which reagents do you routinely include in your penicillin allergy workup? The overwhelming burden of penicillin "allergy" labels remains a global public health concern associated with a myriad of adverse clinical outcomes.[1] The epidemiology and sensitization patterns of penicillin allergy varies greatly and remain largely unknown in the Asia-Pacific (AP) region.[[2], [4]] This international survey was performed to investigate the epidemiology, healthcare infrastructures and clinical practices pertaining to penicillin allergy in AP. In addition to penicillin allergy labels, what proportion (%) of patients have other drug allergy labels?. [Extracted from the article]

An abstract of the article "ASCIA Consensus Statement for Assessment of Immediate (IgE Mediated) Penicillin Allergy," by Michaela Lucas and colleagues is presented.

They subsequently safely administered their second COVID-19 vaccination after basophil activation testing with one child suffering mild urticaria after the challenge.[1] The present recommendations suggest that patients who experience immediate allergic reactions to mRNA COVID-19 vaccines should undergo skin testing for the vaccine or its excipients. Over 13 billion Coronavirus disease 2019 (COVID-19) vaccines have been administered worldwide. Diagnostic accuracy of vaccine and vaccine excipient testing in the setting of allergic reactions to COVID-19 vaccines: a systematic review and meta-analysis. [Extracted from the article]

Background: With the emergence of novel vaccines and new applications for older vaccines, co-administration is increasingly likely. The immunomodulatory effects of BCG could theoretically alter the reactogenicity of co-administered vaccines. Using active surveillance in a randomised controlled trial, we aimed to determine whether co-administration of BCG vaccination changes the safety profile of influenza vaccination. Methods: Participants who received influenza vaccine alone (Influenza group) were compared with those who also received BCG-Denmark vaccine in the contralateral arm (Influenza+BCG group). Data on the influenza vaccination site were collected using serial questionnaires and active follow-up for 3 months post vaccination. Results: Of 1351 participants in the Influenza+BCG group and 1418 participants in the Influenza group, 2615 (94%) provided influenza vaccine safety data. There was no significant difference in the proportion of participants with any local adverse reaction between the Influenza+BCG group and the Influenza group (918/1293 [71.0%] versus (906/1322 [68.5%], p = 0.17). The proportion of participants reporting any pain, erythema and tenderness at the influenza vaccination site were similar in both groups. Swelling was less frequent (81/1293 [6.3%] versus 119/1322 (9.0%), p = 0.01) and the maximal diameter of erythema was smaller (mean 1.8 cm [SD 2.0] versus 3.0 cm [SD 2.5], p<0.001) in the Influenza+BCG group. Sixteen participants reported serious adverse events: 9 participants in the Influenza+BCG group and 7 in the Influenza group. Conclusions: Adverse events following influenza vaccination are not increased when BCG is co-administered. [ABSTRACT FROM AUTHOR]

Background: Although common, antimicrobial allergy labels (AAL) rarely reflect immunologically‐mediated hypersensitivity and can lead to poorer outcomes from alternative antimicrobial agents. Antimicrobial stewardship programs are ideally placed to assess AAL early as a means of improving antimicrobial use. Aims: To quantify the prevalence of AAL in patients referred for antimicrobial stewardship review and assess their impact on antibiotic prescribing, patient mortality, hospital length of stay, readmission and rates of multidrug‐resistant infections. Methods: We conducted a retrospective analysis of adult patients referred for inpatient antimicrobial prospective audit and feedback rounds (PAFR) through an electronic referral system (eReferrals) over a 12‐month period in 2015. Outcome data were collected for a period of 36 months following the initial review. Results: Of the 639 patient records reviewed, 630 met inclusion criteria; 103 (16%) had an AAL, of which 82 (13%) had reported allergies to β‐lactam antibiotics. Those with AAL were significantly less likely to be receiving guideline‐recommended antimicrobial therapy (50% vs 64%, P = 0.0311); however, there were no significant difference in mortality, hospital length of stay, readmission or increased incidence of multidrug‐resistant infections. Conclusions: Our cohort demonstrated that AAL was associated with reduced adherence to antibiotic guidelines. The lack of association with adverse outcomes may reflect limitations within the study including retrospective cohort study numbers and observational nature, further skewed by high rates of poor documentation. A clear opportunity exists for antimicrobial stewardship programs to incorporate allergy assessment, de‐labelling, challenge and referral into these rounds. [ABSTRACT FROM AUTHOR]

A 31-year old man presented with swelling and purpura of the dorsum of the hands following sun exposure. There was a preceding lifelong history of photosensitivity, but this episode, which occurred after the recent commencement of oral iron therapy, and after recent alcohol ingestion, was much more severe than any preceding episode. Skin biopsy performed 48 h after the onset of symptoms showed features consistent with the early stages of leukocytoclastic vasculitis. Direct immunofluorescence showed homogeneous thick staining of the vessel walls with IgG, IgM and IgA, together with abundant perivascular fibrinogen. A subsequent periodic acid-Schiff ( PAS) stain on the skin biopsy revealed thickening of the walls of dermal vessels, which was not discernible in routinely stained (hematoxylin/eosin) sections. The diagnosis of erythropoietic protoporphyria ( EPP) was confirmed by significantly elevated erythrocyte protoporphyrin levels and positive plasma fluorimetry. The diagnosis of porphyria may have been missed by routine skin microscopy if not for the additional information provided by clinical history, direct immunofluorescence and PAS stain. The pathogenesis and histopathology of acute and chronic vascular changes in EPP are reviewed. [ABSTRACT FROM AUTHOR]

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7[sup+]CD45RA[sup-]CD27[sup+]) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vβ repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases. [ABSTRACT FROM AUTHOR]

Containment of hepatitis C virus (HCV) and other chronic human viral infections is associated with persistence of virus-specific CD4 T cells, but ex vivo characterization of circulating CD4 T cells has not been achieved. To further define the phenotype and function of these cells, we developed a novel approach for the generation of tetrameric forms of MHC class II/peptide complexes that is based on the cellular peptide-exchange mechanism. HLA-DR molecules were expressed as precursors with a covalently linked CLIP peptide, which could be efficiently exchanged with viral peptides following linker cleavage. In subjects who spontaneously resolved HCV viremia, but not in those with chronic progressive infection, HCV tetramer-labeled cells could be isolated by magnetic bead capture despite very low frequencies (1:1,200 to 1:111,000) among circulating CD4 T cells. These T cells expressed a set of surface receptors (CCR7+CD45RA-CD27+) indicative of a surveillance function for secondary lymphoid structures and had undergone significant in vivo selection since they utilized a restricted Vbeta repertoire. These studies demonstrate a relationship between clinical outcome and the presence of circulating CD4 T cells directed against this virus. Moreover, they show that rare populations of memory CD4 T cells can be studied ex vivo in human diseases. [ABSTRACT FROM AUTHOR]

Objectives: Idiopathic mast cell disorders, a recently defined and recognised syndrome in clinical practice, are similar to the previously termed non‐clonal mast cell disorder. Patients with idiopathic mast cell activation syndrome (MCAS) suffer all the classical signs of mast cell activation but do not have evidence of mast cell clonality. Furthermore, treatment of these patients can be limited and burdensome in those with refractory symptoms. Methods: Here, we describe treatment of a patient with idiopathic MCAS utilising a single monthly subcutaneous injection of omalizumab and review the current classification and therapeutic options for clonal and non‐clonal MCAS. Results: Low‐dose omalizumab treatment has successfully led to a 5‐year, sustained clinical response, controlled debilitating symptoms of mast cell activation and allowed for reintroduction and long‐term maintenance of bee venom subcutaneous immunotherapy. Conclusion: Low‐dose omalizumab of 150 mg monthly should be considered for maintenance management of patients with idiopathic MCAS for its cost and quality‐of‐life benefits. [ABSTRACT FROM AUTHOR]

Objectives: The epidemiology, clinical characteristics and outcomes of antimicrobial-associated anaphylaxis remain ill-defined. We sought to examine antimicrobial anaphylaxis with regard to: (i) the frequency of implicated antimicrobials; (ii) attributable mortality; and (iii) referral for definitive allergy assessment.Methods: This was conducted through a national retrospective multicentre cohort study at five Australian tertiary hospitals (January 2010 to December 2015). Cases of antimicrobial anaphylaxis were identified from ICD-10 coding and adverse drug reaction committee databases.Results: There were 293 participants meeting the case definition of antimicrobial anaphylaxis and 310 antimicrobial anaphylaxis episodes. Of 336 implicated antimicrobials, aminopenicillins (62/336, 18.5%) and aminocephalosporins (57/336, 17%) were implicated most frequently. ICU admission occurred in 43/310 (13.9%) episodes; however, attributable mortality was low (3/310, 1%). The rate of anaphylaxis to IV antibiotics was 3.5 (95% CI=2.9-4.3) per 100 000 DDDs and the rate of hospital-acquired anaphylaxis was 1.9 (95% CI=2.1-3.3) per 100 000 occupied bed-days. We observed overall low rates of hospital discharge documentation (222/310, 71.6%) and follow-up by specialist allergy services (73/310, 23.5%), which may compromise medication safety and antimicrobial prescribing in future.Conclusions: This study demonstrated that a high proportion of severe immediate hypersensitivity reactions presenting or acquired in Australian hospitals are secondary to aminopenicillins and aminocephalosporins. Overall rates of hospital-acquired anaphylaxis, predominantly secondary to cephalosporins, are low, and also associated with low inpatient mortality. [ABSTRACT FROM AUTHOR]

Human immunodeficiency virus (HIV) can adapt to an individual's T cell immune response via genomic mutations that affect antigen recognition and impact disease outcome. These viral adaptations are specific to the host's human leucocyte antigen (HLA) alleles, as these molecules determine which peptides are presented to T cells. As HLA molecules are highly polymorphic at the population level, horizontal transmission events are most commonly between HLA-mismatched donor/recipient pairs, representing new immune selection environments for the transmitted virus. In this study, we utilised a deep sequencing approach to determine the HIV quasispecies in 26 mother-to-child transmission pairs where the potential for founder viruses to be pre-adapted is high due to the pairs being haplo-identical at HLA loci. This scenario allowed the assessment of specific HIV adaptations following transmission in either a non-selective immune environment, due to recipient HLA mismatched to original selecting HLA, or a selective immune environment, mediated by matched donor/recipient HLA. We show that the pattern of reversion or fixation of HIV adaptations following transmission provides insight into the replicative cost, and likely compensatory networks, associated with specific adaptations in vivo. Furthermore, although transmitted viruses were commonly heavily pre-adapted to the child's HLA genotype, we found evidence of de novo post-transmission adaptation, representing new epitopes targeted by the child's T cell response. High-resolution analysis of HIV adaptation is relevant when considering vaccine and cure strategies for individuals exposed to adapted viruses via transmission or reactivated from reservoirs. Author summary: Highly mutable pathogens utilise genetic variations within T cell epitopes as a mechanism of immune escape (viral adaptation). The diversity of the human leucocyte antigen (HLA) molecules that present viral targets to T cells in human populations partially protects against rapid population-level accumulation of human immunodeficiency virus (HIV) adaptations through horizontal transmissions. In contrast, vertical transmissions occur between haplo-identical mother/child pairs, and potentially include adaptive changes through father-mother-child transmission, representing a pathway to complete pre-adaptation to HLA alleles in child hosts over only two transmission events. We utilised next-generation sequencing to examine HIV evolution in the unique setting of vertical HIV transmission. We predict the in vivo replicative cost and immune benefit of specific HIV adaptations that could be used to inform vaccine design and cure strategies to combat viral immune adaptation. [ABSTRACT FROM AUTHOR]

Aim: To determine if skin testing (ST) in addition to extended oral provocation challenge (OPC) is necessary for beta-lactam allergy verification in an Australian paediatric population.Methods: This was a retrospective study (176 children) that undertook assessments for beta-lactam allergy from 2006 to 2015 at a tertiary paediatric hospital. Patients either underwent direct OPC without ST or ST plus challenge if ST was negative.Results: The analysis included children with a history of varying rash types/severity as well as angioedema and reported anaphylaxis. A direct OPC was undertaken in 73 children. Three children reacted with one anaphylaxis. A total of 103 children underwent ST, with 13 children (12.6%) reacting. Of the 90 who subsequently proceeded to OPC, 4 reacted. A total of 132 children were given an extended oral course of the culprit antibiotic, to which 6 children reacted.Conclusions: A direct OPC with the culprit drug in Australian children can be safely performed, avoiding resource-intensive and painful ST. Our data demonstrate that a prior history of anaphylaxis does not necessarily predict IgE-mediated allergy, as detected by positive immediate ST or reactions to oral challenge. Such history should not detract from efforts to assess these children for antibiotic allergy. We suggest that extended courses of at least 5 days are important in paediatric antibiotic de-labelling as six children (4.5% of those who were prescribed the extended course) reacted in our study and even developed symptoms late in the extended course, from days 2 to 6. [ABSTRACT FROM AUTHOR]

Summary: Background and aim: Pediatric patients increasingly report allergies, including allergies to food and medications. We sought to determine the incidence and, nature of parent‐reported allergies in children presenting for surgery and its significance for anesthetists. Methods: We prospectively collected data on admissions through our surgical admission unit over a 2‐month period at a pediatric tertiary care teaching hospital. Data collected included patient demographics, history of atopy, with more comprehensive information collected if an allergy was reported. A clinical immunologist and an anesthetist reviewed the documentation of all patients reporting an allergy. Results: We reviewed 1001 pediatric patients, 158 (15.8%) patients with parent‐reported allergies; to medications/drugs (n = 73), food (n = 66), environmental allergens (dust/grasses, n = 35), tapes/dressings (n = 27), latex (n = 4), and venom (eg, bee, wasp, n = 9). Forty‐one patients reported antibiotic allergies, with Beta‐lactam antibiotics being the most common, with the majority presenting with rash alone (57%). Ten patients reported allergies to nonsteroidal anti‐inflammatory drugs and eight to opioids. Twenty‐four patients reported egg and/or peanut allergy. Only 3/1001 (0.3%) patients were deemed to have evidence of likely IgE‐mediated drug allergy. Of the reported allergies, only 60 (38.2%) had been investigated prior, most likely to be followed up were food (53%) and environmental allergies (44.4%). Only 4/73 (5.5%) reported medication allergies had further follow‐up. Just four patients (0.4% of the entire cohort) had drug sensitivities/allergies that were likely to majorly alter anesthesia practice. Conclusion: Only the minority of parent‐reported allergies in pediatric surgical patients were specialist confirmed and likely to be clinically relevant. Self‐reported food allergy is commonly specialist verified whereas reactions to medications were generally not. Over‐reporting of allergies is increasingly common and limits clinician choice of medications. Better education of patients and their families and more timely verification or dismissal of parent‐reported reactions is urgently needed. [ABSTRACT FROM AUTHOR]

Abstract Objective To evaluate the prevalence of anti-AQP4 antibody in serum and CSF samples from patients being investigated for possible neuromyelitis optica spectrum disorder (NMOSD) referred to the PathWest State reference laboratory using a sensitive cell-based assay (CBA). Background NMOSD is an inflammatory CNS disease distinct from MS, which is relatively rare in Western countries. A proportion of patients with NMOSD have detectable serum IgG antibodies that target the water channel aquaporin-4 (AQP4-IgG), but the frequency varies in different populations studied and according to the assay method employed. Methods Sera or CSF from a diagnostic cohort of 196 consecutive patients with possible NMOSD which had previously been screened by indirect immunofluorescence (IIF) on primate cerebellum were re-tested for AQP4-IgG reactivity to the M1 and M23 isoforms of AQP4 using a commercial CBA. A control group of 205 patients with definite MS was also included in the study. Results Of the 196 patients, only 5 sera were AQP4-IgG positive, representing 2.6% of patients in the diagnostic cohort. All 5 AQP4-IgG positive patients fulfilled the 2015 revised diagnostic criteria for NMOSD and were females of varied ethnic origins, 4 of whom had longitudinally extensive transverse myelitis. The CBA confirmed AQP4-IgG positivity in the four patients previously reported as positive by IIF, and an additional patient with NMOSD who had previously been diagnosed as MS was also identified. None of the 205 MS sera were AQP4-IgG positive. Conclusions Our study confirms the utility and greater reliability of the M1/M23 CBA for detecting AQP4-IgG in patients with possible NMOSD, and indicates a prevalence of seropositive NMOSD in the Western Australian population similar to that in other Western populations. Graphical abstract Unlabelled Image Highlights • The frequency of AQP4-IgG in Western Australian patients with possible NMOSD was 2.6%. • All AQP4-IgG positive cases were females of varied ethnic background. • The cell-based assay has a greater reliability than tissue-based IIF for detecting AQP4-IgG in cases with possible NMOSD. • The prevalence of seropositive NMOSD in Western Australia is similar to that in other Western populations. [ABSTRACT FROM AUTHOR]

Advances in reproductive medicine have significantly increased the success of fertility treatments. Nevertheless, some women experience recurrent implantation failure (RIF) after in‐vitro fertilization (IVF) or recurrent pregnancy loss (RPL). Imbalances in the immune system and failure to achieve immune tolerance to the foetus have been implicated as potentially modifiable causes of idiopathic RIF and RPL. As such, women are increasingly being treated with immunomodulatory agents in an attempt to achieve a successful pregnancy. This systematic review examines the published evidence on immune changes in these patients, the use of immunomodulation therapies and diagnostic testing modalities to guide their use or to identify patient subsets most likely to benefit. The PubMed database was searched for the terms “recurrent implantation failure” and “recurrent pregnancy loss” in conjunction with T‐helper (Th) cells and their subsets in particular; Th1, Th2, Th17 and T‐regulatory (Treg) cells, natural killer (NK) cells, cytokine imbalance as well as immune modulators and immune suppressants. The reference lists of articles were examined to identify additional articles. There remains limited data on the immunological changes in cytokine and cellular profiles during the hormonal cycle as well as prior to, during and after implantation in health as well as idiopathic RIF and RPL. There is a need to advance immunological diagnostics to match the clinical need in this emerging field and to guide clinicians to make optimal and safe therapeutic choices. It is also imperative that the well‐being of the infants conceived after such intervention is monitored. [ABSTRACT FROM AUTHOR]

Non-immediate allergic cutaneous reactions to heparins have been increasingly reported, typically manifesting as large, eczematous plaques at sites of subcutaneous injection. Patients may demonstrate cross-reactivity between unfractionated heparin, low molecular weight heparin and semi-synthetic heparinoids, making finding an alternative difficult. Fondaparinux has been identified as a useful alternative in such patients; here we present the first two documented cases in Australia and a literature review. [ABSTRACT FROM AUTHOR]

An abstract of the article "Glucocorticoids: From Hero to Villain," by Dinusha Chandratilleke and colleagues is presented.

Objective: Developing a vaccine that is cross-reactive between HCV genotypes requires data on T cell antigenic targets that extends beyond genotype-1. We characterised T cell immune responses against HCV genotype-3, the most common infecting genotype in the UK and Asia, and assessed within genotype and between genotype cross-reactivity. Design: T cell targets were identified in 140 subjects with either acute, chronic or spontaneously resolved HCV genotype-3 infection using (1) overlapping peptides and (2) putative human leucocyte antigens (HLA)-class-I wild type and variant epitopes through the prior assessment of polymorphic HCV genomic sites associated with host HLA, in IFN-ELISpot assays. CD4 +/CD8+ T cell subsets were defined and viral variability at T cell targets was determined through population analysis and viral sequencing. T cell cross-reactivity between genotype-1 and genotype-3 variants was assessed. Results: In resolved genotype-3 infection, T cells preferentially targeted non-structural proteins at a high magnitude, whereas in chronic disease T cells were absent or skewed to target structural proteins. Additional responses to wild type but not variant HLA predicted peptides were defined. Major sequence viral variability was observed within genotype-3 and between genotypes 1 and 3 HCV at T cell targets in resolved infection and at dominant epitopes, with limited T cell cross-reactivity between viral variants. Overall 41 CD4/CD8+ genotype- 3 T cell targets were identified with minimal overlap with those described for HCV genotype-1. Conclusions: HCV T cell specificity is distinct between genotypes with limited T cell cross-reactivity in resolved and chronic disease. Therefore, viral regions targeted in natural HCV infection may not serve as attractive targets for a vaccine that aims to protect against multiple HCV genotypes. [ABSTRACT FROM AUTHOR]

Interstitial lung disease (ILD) is a significant complication of Sjögren syndrome (SS) associated with increased morbidity and mortality. The mainstay of treatment remains corticosteroid administration, with or without additional immunosuppressive therapies. Preliminary studies in SS have shown benefit in glandular and serologic parameters following treatment with the CTLA4 immunoglobulin fusion protein abatacept. Topical tacrolimus has been effective for ocular symptoms in SS, but systemic therapy has not been reported. We describe the first case, to our knowledge, of the successful use of a combination of systemic tacrolimus and abatacept in severe refractory SS and related ILD. [ABSTRACT FROM AUTHOR]

Characterisation of Hepatitis C virus (HCV)-specific CD8+ T-cell responses in the context of multiple HCV exposures is critical to identify broadly protective immune responses necessary for an effective HCV vaccine against the different HCV genotypes. However, host and viral genetic diversity complicates vaccine development. To compensate for the observed variation in circulating autologous viruses and host molecules that restrict antigen presentation (human leucocyte antigens; HLA), this study used a reverse genomics approach that identified sites of viral adaptation to HLA-restricted T-cell immune pressure to predict genotype-specific HCV CD8+ T-cell targets. Peptides representing these putative HCV CD8+ T-cell targets, and their adapted form, were used in individualised IFN-γ ELISpot assays to screen for HCV-specific T-cell responses in 133 HCV-seropositive subjects with high-risk of multiple HCV exposures. The data obtained from this study i) confirmed that genetic studies of viral evolution is an effective approach to detect novel in vivo HCV T-cell targets, ii) showed that HCV-specific T-cell epitopes can be recognised in their adapted form and would not have been detected using wild-type peptides and iii) showed that HCV-specific T-cell (but not antibody) responses against alternate genotypes in chronic HCV-infected subjects are readily found, implying clearance of previous alternate genotype infection. In summary, HCV adaptation to HLA Class I-restricted T-cell responses plays a central role in anti-HCV immunity and multiple HCV genotype exposure is highly prevalent in at-risk exposure populations, which are important considerations for future vaccine design. [ABSTRACT FROM AUTHOR]

Since the discovery of induced pluripotent stem cells (iPSC) in 2006, the symptoms of many human diseases have been reversed in animal models with iPSC therapy, setting the stage for future clinical development. From the animal data it is clear that iPSC are rapidly becoming the lead cell type for cell replacement therapy and for the newly developing field of iPSC-derived body organ transplantation. The first human pathology that might be treated in the near future with iPSC is age-related macular degeneration (AMD), which has recently passed the criteria set down by regulators for phase I clinical trials with allogeneic human embryonic stem cell-derived cell transplantation in humans. Given that iPSC are currently in clinical trial in Japan (RIKEN) to treat AMD, the establishment of a set of international criteria to make clinical-grade iPSC and their differentiated progeny is the next step in order to prepare for future autologous cell therapy clinical trials. Armed with clinical-grade iPSC, we can then specifically test for their threat of cancer, for proper and efficient differentiation to the correct cell type to treat human disease and then to determine their immunogenicity. Such a rigorous approach sets a far more relevant paradigm for their intended future use than non-clinical-grade iPSC. This review focuses on the latest developments regarding the first possible use of iPSC-derived retinal pigment epithelial cells in treating human disease, covers data gathered on animal models to date and methods to make clinical-grade iPSC, suggests techniques to ensure quality control and discusses possible clinical immune responses. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV)-specific CD8+ T cells in persistent HCV infection are low in frequency and paradoxically show a phenotype associated with controlled infections, expressing the memory marker CD127. We addressed to what extent this phenotype is dependent on the presence of cognate antigen. We analyzed virus-specific responses in acute and chronic HCV infections and sequenced autologous virus. We show that CD127 expression is associated with decreased antigenic stimulation after either viral clearance or viral variation. Our data indicate that most CD8 T-cell responses in chronic HCV infection do not target the circulating virus and that the appearance of HCV-specific CD127+ T cells is driven by viral variation. [ABSTRACT FROM AUTHOR]

Cellular immune responses during acute Hepatitis C virus (HCV) and HIV infection are a known correlate of infection outcome. Viral adaptation to these responses via mutation(s) within CD8+ T-cell epitopes allows these viruses to subvert host immune control. This study examined HCV evolution in 21 HCV genotype 1-infected subjects to characterise the level of viral adaptation during acute and early HCV infection. Of the total mutations observed 25% were within described CD8+ T-cell epitopes or at viral adaptation sites. Most mutations were maintained into the chronic phase of HCV infection (75%). The lack of reversion of adaptations and high proportion of silent substitutions suggests that HCV has structural and functional limitations that constrain evolution. These results were compared to the pattern of viral evolution observed in 98 subjects during a similar phase in HIV infection from a previous study. In contrast to HCV, evolution during acute HIV infection is marked by high levels of amino acid change relative to silent substitutions, including a higher proportion of adaptations, likely reflecting strong and continued CD8+ T-cell pressure combined with greater plasticity of the virus. Understanding viral escape dynamics for these two viruses is important for effective T cell vaccine design. [ABSTRACT FROM AUTHOR]

Background: Cellular immunity plays a crucial role in cytomegalovirus (CMV) infection and substantial populations of CMV-specific T cells accumulate throughout life. However, although CMV infection occurs during childhood, relatively little is know about the typical quantity and quality of T cell responses in pediatric populations. Methods: One thousand and thirty-six people (Male/Female = 594/442, Age: 0-19 yr.; 959 subjects, 20-29 yr.; 77 subjects) were examined for HLA typing. All of 1036 subjects were tested for HLA-A2 antigen. Of 1036 subjects, 887 were also tested for HLA-A23, 24 antigens. In addition, 50 elderly people (Male/Female = 11/39, Age: 60-92 yr.) were also tested for HLA-A2 antigen. We analyzed the CD8+ T cell responses to CMV, comparing these to responses in children and young. The frequencies, phenotype and function CD8+ T cells for two imunodominant epitopes from pp65 were measured. Results: We observed consistently high frequency and phenotypically "mature" (CD27 low, CD28 low, CD45RA+) CMV-specific CD8+ T cell responses in children, including those studied in the first year of life. These CD8+ T cells retained functionality across all age groups, and showed evidence of memory "inflation" only in later adult life. Conclusion: CMV consistently elicits a very strong CD8+ T cell response in infants and large pools of CMV specific CD8+ T cells are maintained throughout childhood. The presence of CMV may considerably mould the CD8+ T cell compartment over time, but the relative frequencies of CMV-specific cells do not show the evidence of a population-level increase during childhood and adulthood. This contrast with the marked expansion ("inflation") of such CD8+ T cells in older adults. This study indicates that large scale analysis of peptide specific T cell responses in infants is readily possible. The robust nature of the responses observed suggests vaccine strategies aimed at priming and boosting CD8+ T cells against major pathogens (including HIV, malaria and CMV itself) could be successful in this age-group. [ABSTRACT FROM AUTHOR]

The basis of chronic infection following exposure to hepatitis C virus (HCV) infection is unexplained. One factor may be the low frequency and immature phenotype of virus-specific CD8+ T cells. The role of CD4+CD25+ T regulatory (Treg) cells in priming and expanding virus-specific CD8+ T cells was investigated. Twenty HLA-A2-positive patients with persistent HCV infection and 46 healthy controls were studied. Virus-specific CD8+ T-cell proliferation and gamma interferon (IFN-γ) frequency were analyzed with/without depletion of Treg cells, using peptides derived from HCV, Epstein-Barr virus (EBV), and cytomegalovirus (CMV). CD4+CD25+ Treg cells inhibited anti-CD3/CD28 CD8+ T-cell proliferation and perforin expression. Depletion of CD4+CD25+ Treg cells from chronic HCV patients in vitro increased HCV and EBV peptide-driven expansion (P = 0.0005 and P = 0.002, respectively) and also the number of HCV- and EBV-specific IFN-γ-expressing CD8+ T cells. Although stimulated CD8+ T cells expressed receptors for transforming growth factor beta and interleukin-10, the presence of antibody to transforming growth factor beta and interleukin-10 had no effect on the suppressive effect of CD4+CD25+ regulatory T cells on CD8+ T-cell proliferation. In conclusion, marked CD4+CD25+ regulatory T-cell activity is present in patients with chronic HCV infection, which may contribute to weak HCV-specific CD8+ T-cell responses and viral persistence. [ABSTRACT FROM AUTHOR]

HIV-specific CD4+ T helper lymphocytes are preferred targets for infection. Although complete interruption of combination antiretroviral therapy (ART) can form part of therapeutic manipulations, there is grave concern that the resumption of viral replication might destroy, perhaps irreversibly, these T helper populations. High viremia blocks the proliferation capacity of HIV-specific helper cells. However, cytokine production assays imply that some antigen-specific effector function is retained. Despite this careful work, it remains unclear whether the return of HIV-1 replication physically destroys HIV-1-specific T helper cells in the peripheral blood. Difficulties in producing stable peptide-MHC class II complexes and the very low frequencies of antigen-specific CD4+ T cells have delayed the application of this powerful technique. Here we employ HLA class II tetramers and validate a sensitive, quantitative cell-enrichment technique to detect HIV-1 T helper cells. We studied patients with early-stage HIV infection who were given a short, fixed course of ART as part of a clinical study. We did not find significant deletion of these cells from the peripheral circulation when ART was stopped and unfettered HIV replication returned. The turnover of these virus-specific cells increased and they adopted an effector phenotype when viremia returned. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to the article "A Prospective Large-Scale Study of Methods for the Detection of Latent Mycobacterium Tuberculosis Infection in Refugee Children" by M. Lucas et al.

An abstract of the article "Assessing the Utility of the Basophil Activation test for the Diagnosis of Suspected Anaesthetic or Antibiotic Allergy in a Western Australian Cohort," by Joel Kidman and colleagues is presented.

An abstract of the article "Increase, Characteristics, Identification and Management of Anaphylaxix: A Perspective of an Australian Emergency Department," by Kristina Rueter and colleagues is presented.

An abstract of the article "Fondaparinux as an Alternative in Non-Immediate Hypersensitivity to Heeparins and Heparinoids," by Grace Thompson, and colleagues is presented.

Hepatitis C virus subtype 3a is a highly prevalent and globally distributed strain that is often associated with infection via injection drug use. This subtype exhibits particular phenotypic characteristics. In spite of this, detailed genetic analysis of this subtype has rarely been performed. We performed full-length viral sequence analysis in 18 patients with chronic HCV subtype 3a infection and assessed genomic viral variability in comparison to other HCV subtypes. Two novel regions of intragenotypic hypervariability within the envelope protein E2, of HCV genotype 3a, were identified. We named these regions HVR495 and HVR575. They consisted of flanking conserved hydrophobic amino acids and central variable residues. A 5-amino-acid insertion found only in genotype 3a and a putative glycosylation site is contained within HVR575. Evolutionary analysis of E2 showed that positively selected sites within genotype 3a infection were largely restricted to HVR1, HVR495, and HVR575. Further analysis of clonal viral populations within single hosts showed that viral variation within HVR495 and HVR575 were subject to intrahost positive selecting forces. Longitudinal analysis of four patients with acute HCV subtype 3a infection sampled at multiple time points showed that positively selected mutations within HVR495 and HVR575 arose early during primary infection. HVR495 and HVR575 were not present in HCV subtypes 1a, 1b, 2a, or 6a. Some variability that was not subject to positive selection was present in subtype 4a HVR575. Further defining the functional significance of these regions may have important implications for genotype 3a E2 virus-receptor interactions and for vaccine studies that aim to induce cross-reactive anti-E2 antibodies. [ABSTRACT FROM AUTHOR]