Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9.

BACKGROUND: Mucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917. RESULTS: The HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) - rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu. CONCLUSIONS: The CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

Pregnancy outcome in women with gestational diabetes - A longitudinal study of changes in demography and treatment modalities.

INTRODUCTION: Gestational diabetes is on the rise and demographics are changing in many countries due to increased migration. Simultaneously, the treatment of gestational diabetes in our clinic has shifted towards metformin with substantially less insulin treatment. The aim was to study the impact of these changes on metabolic control and pregnancy outcome by comparing women diagnosed with gestational diabetes during 2012-2013 and 2016-2017. MATERIAL AND METHODS: Our universal Oral Glucose Tolerance Test screening program for gestational diabetes diagnosed 199 women with singleton pregnancies during 2012-2013 and 203 during 2016-2017. Treatment and achieved metabolic control in the two different time periods were compared. Pregnancy outcome data related to gestational diabetes were retrieved from case notes and compared between the different time periods. RESULTS: When comparing results from 2016-2017 with 2012-2013 there was no difference in maternal weight or weight gain. There was a higher frequency of heredity (52.6 vs 35.4%; P = 0.001) and non-Scandinavian ethnicity (46.5 vs 33.8%; P = 0.011).The frequency of smoking during pregnancy was significantly lower (2.6 vs 7.7%; P = 0.023) There was an improved metabolic control as measured by median glucose in 2016-2017 compared with 2012-2013 (5.8 vs 6.2 mmol/L; P < 0.001). Insulin was less frequently used in 2016-2017 than in 2012-2013 (32.5 vs 44.7%; P = 0.012). There was a significant increase in the use of metformin (14.8 vs 0%; P < 0.001). There were no differences regarding the frequency of large-for-gestational-age infants (8.2% vs 7.3%; P = 0.762) or macrosomia (16.3 vs 15.1%; P = 0.745), median birthweight (3510 vs 3521; P = 0.879), frequency of cesarean section (28.1 vs 27.8%; P = 0.951) or Apgar scores at 10 minutes (10 [3-10] vs 10 [7-10]; P = 0.290). CONCLUSIONS: In an increasing but changing population of gestational diabetes women in our region, with more hereditary and non-Scandinavian origins, but with fewer smokers, metabolic control has improved with maintained favorable pregnancy outcomes, with more frequent use of metformin and substantially less use of insulin treatment.

Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen.

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gagp37 and two vaccinations with MVA-CMDR encoding subtype A Gagp55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ+) Gag-specific T-cell responses were dominated by CD4+ T cells (P < 0.001 compared to CD8+ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gagp24 regions, more conserved between prime and boost, compared to those of regions within Gagp15 (not primed) and Gagp17 (less conserved; P < 0.0001 for both). Four regions within Gagp24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r2 = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4+ T cells and that targeted multiple antigenic regions within the conserved Gagp24 protein.IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

IgG4 subclass glutamic acid decarboxylase antibodies (GADA) are associated with a reduced risk of developing type 1 diabetes as well as increased C-peptide levels in GADA positive gestational diabetes.

Some women with gestational diabetes (GDM) present with autoantibodies associated with type 1 diabetes. These are usually directed against glutamic acid decarboxylase (GADA) and suggested to predict development of type 1 diabetes. The primary aim of this study was to investigate if GADA IgG subclasses at onset of GDM could assist in predicting postpartum development. Of 1225 women diagnosed with first-time GDM only 51 were GADA-positive. Total GADA was determined using ELISA. GADA subclasses were determined with radioimmunoassay. Approximately 25% of GADA-positive women developed type 1 diabetes postpartum. Titers of total GADA were higher in women that developed type 1 diabetes (142.1 vs 74.2u/mL; p=0.04) and they also had lower titers of GADA IgG4 (index=0.01 vs 0.04; p=0.03). In conclusion we found that that women with high titers of total GADA but low titers of GADA IgG4 were more prone to develop type 1 diabetes postpartum.

Towards normalized birthweight in gestational diabetes mellitus.

INTRODUCTION: The objective was to describe pregnancy outcomes in gestational diabetes mellitus (GDM) in comparison with general population in Sweden. MATERIAL AND METHODS: A population-based retrospective study using University hospital records and Swedish Medical Birth Register was carried out on pregnant women with well-monitored GDM (n = 870) and pregnancies in the Swedish Medical Birth Register (n = 1 553 420). Data from GDM pregnancies was compared to pregnancies in the whole of Sweden during 1995-2010. The main outcome measures were age, first bodyweight in and weight gain during pregnancy, birthweight, gestational length, percentage of cesarean deliveries. RESULTS: First maternal bodyweight during the GDM pregnancy was higher than in the general population, 72.4 ± 17.4 kg (n = 837) vs. 67.3 ± 12.6 kg (n = 1 383 000; p < 0.0001). Women with GDM gained less weight during pregnancy 9.9 ± 5.8 kg (n = 703) compared to the general population, 13.2 ± 5.7 kg (n = 482 860; p < 0.0001). Mean birthweight in GDM pregnancies was 3564 ± 500 g (n = 743) compared to 3580 ± 483 g for the general population (n = 1 316 364; p = ns). Gestational length was slightly shorter, 39.4 weeks in the GDM pregnancies (n = 683) vs. 39.5 weeks (n = 1 319 876; p = 0.02) in the general population and the percentage of cesarean deliveries higher in the GDM pregnancies at 18.4% (n = 712) vs. 13.3% (n = 1 322 242; p < 0.0001). CONCLUSIONS: Though many studies have shown an increased risk of macrosomia in GDM pregnancies, remaining even after ambitious management programs, we show no difference in birthweight. This may be due to a combination of intense efforts to achieve good metabolic control during pregnancy and shorter pregnancy duration. Preventing unduly large babies is crucial to minimize adverse pregnancy outcomes.

Increased risk for overweight among Swedish children born to mothers with gestational diabetes mellitus.

OBJECTIVE: Investigate the effects of maternal gestational diabetes mellitus (GDM) on height, weight, and body mass index (BMI) in offspring compared both to their siblings and to age-specific BMI reference values in Sweden. Their parents present BMI was also investigated. METHODS: The growth of 232 offspring to 110 women with at least one pregnancy with GDM, were studied up to 12 yr of age. Height and weight of children were collected from Health Care Centres and compared to age-specific reference values in Sweden. Self-reported height and weight of the parents were collected at follow-up. RESULTS: For boys, weight was higher at birth and at 8-10 yr of age, giving a higher BMI at 7-10 yr of age. Girls had an accelerated height growth at all ages, combined with an increased weight of varying degree resulting in higher BMI at birth and at 4-12 yr of age. A similar pattern was observed in siblings born after a normal pregnancy. Median BMI of mothers at follow-up was 25.4 (18.3-59.5 n = 105) and 26.5 (18.6-38.1 n = 90) for fathers. CONCLUSIONS: Children born to mothers with prior GDM have a higher risk of overweight and obesity later in life. This is most likely due to life style habits rather than intrauterine factors, as the same BMI pattern was found in siblings born after a normal pregnancy. However, the design of the study could not rule out the role of genetic factors. Priority should be given to early life style intervention in these families.

Cystatin C as an adjunct to HbA1c may prove useful in predicting the development of diabetic complications in children and adolescents with type 1 diabetes.

Purpose: Complications from diabetes mellitus can occur over time and although glycosylated hemoglobin (HbA1c) is a good biomarker for glycaemic control, other factors also contribute to the development of complications in type 1 diabetes. More markers able to identify the risk of complications are needed. This study aimed to investigate plasma levels of FGF21, Cystatin C, lipocalin-2, and MMP-9 in children and adolescents with different duration of type 1 diabetes and possible correlation to HbA1c to identify potential biomarkers of future complication development. Methods: Patients (n = 244, 0-18 years) with type 1 diabetes, at Helsingborg's Hospital, Sweden, were included in this study. Circulating levels of FGF21, Cystatin C, lipocalin-2, and MMP-9 were investigated in plasma using automated ELISA with the ELLA™ system and standardised controls. Results: Cystatin C levels were elevated in patients with diabetes duration longer than 5 years (P < 0.001). HbA1c and Cystatin C levels were inversely correlated for all participants (rs = - 0.23, CI95: -0.35--0.10; P < 0.001). A stepwise multiple regression analysis showed that HbA1c (P < 0.001) and Cystatin C (P = 0.03) were associated to the duration of diabetes at sampling while MMP-9, lipocalin-2, and FGF21 did not reach statistical significance. Conclusion: In conclusion, Cystatin C levels were higher in patients with diabetes duration longer than 5 years, and inverse correlation was found between HbA1c and Cystatin C levels as well as duration of diabetes. Cystatin C may prove useful as an adjunct to HbA1c in predicting eventual development of diabetic complications. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-024-01419-1.

Are soluble E-selectin, ICAM-1, and VCAM-1 potential predictors for the development of diabetic retinopathy in young adults, 15-34 years of age? A Swedish prospective cohort study.

The aim of this study was to determine plasma levels of three adhesion molecules that may contribute to the development of diabetic retinopathy; soluble endothelial selectin (sE-selectin), soluble intercellular adhesion molecule-1 (sICAM-1), and soluble vascular cell adhesion molecule-1 (sVCAM-1), in young adults, aged 15-34 years at diagnosis of diabetes, to find potential predictors for development of retinopathy, and to evaluate their relation to diabetes associated autoantibodies. Participants with type 1 (n = 169) and type 2 diabetes (n = 83) were selected from the complications trial of the Diabetes Incidence Study in Sweden and classified in two subgroups according to presence (n = 80) or absence (n = 172) of retinopathy as determined by retinal photography at follow-up 8-10 years after diagnosis of diabetes. Blood samples were collected at diagnosis in 1987-88. The levels of sE-selectin, sICAM-1, and sVCAM-1 were analysed by enzyme-linked immunosorbent assay and islet cell antibodies by a prolonged two-colour immunofluorescent assay. Mean HbA1c (p<0.001) and clinical characteristics: mean body mass index (p = 0.019), systolic blood pressure (p = 0.002), diastolic blood pressure (p = 0.003), male gender (p = 0.026), and young age at diagnosis of diabetes (p = 0.015) remained associated with development of retinopathy in type 1 diabetes. However, in a multivariate analysis only HbA1c remained as a risk factor. sE-selectin was significantly higher in the group with type 2 diabetes and retinopathy, compared to the group with type 2 diabetes without retinopathy (p = 0.04). Regarding sE-selectin, sICAM-1, and sVCAM-1 in participants with type 1 diabetes, no differences were observed between the groups with or without retinopathy. This trial confirmed the role of HbA1c and clinical characteristics as predictors for development of retinopathy in type 1 diabetes. sE-selectin stands out as a potential predictor for development of retinopathy in type 2 diabetes, whereas a predictive role for sICAM-1 and sVCAM-1 could not be identified neither for type 1 nor type 2 diabetes.

Laboratory-based evaluation of the 4th-generation AlereTM HIV Combo rapid point-of-care test.

BACKGROUND: Mozambique is a high-prevalence country for HIV and early detection of new HIV infections is crucial for control of the epidemic. We aimed to evaluate the accuracy of the 4th-generation rapid diagnostic test (RDT) AlereTM HIV Combo in detecting acute and seroconverted HIV-infection, among sexually-active women attending three clinical health centers in Maputo, Mozambique. METHODS: Women aged 14-55 years (n = 920) seeking care at the Mavalane Health Area, Maputo (February 2018-January 2019) were included, and blood specimens sampled. Sociodemographic and sexual behavior data were collected. Point-of-care HIV testing was performed using Alere DetermineTM HIV-1/2 and Uni-GoldTM HIV-1/2. All samples were also tested using Enzygnost® HIV Integral 4 and Innotest® HIV Antigen mAb in laboratory. The 4th-generation RDT AlereTM HIV Combo was evaluated on serum samples in the laboratory. Finally, Innotest® HIV Antigen mAb, Enzygnost® HIV Integral 4 (Ag/Ab), and HIV RNA quantification acted as gold standard assays in the evaluation of AlereTM HIV Combo test for HIV antigen detection (in clinical samples and in three HIV-1 seroconversion panels). RESULTS: The antibody component of the 4th generation AlereTM HIV Combo RDT demonstrated a sensitivity and specificity of 100% examining clinical samples. However, the test did not detect HIV p24 antigen in any clinical samples, while Innotest® HIV Antigen mAb, verified by Enzygnost® HIV Integral 4 (Ag/Ab) and/or HIV RNA quantification, detected HIV antigen in six clinical samples. Furthermore, the AlereTM HIV Combo RDT had a low sensitivity in the detection of HIV p24 antigen in seroconversion panels. The HIV prevalence among the examined women was 17.8%. CONCLUSIONS: The 4th-generation RDT AlereTM HIV Combo showed similar sensitivity to the 3rd-generation RDTs to detect seroconverted HIV-infections. However, the sensitivity for detection of HIV p24 antigen and diagnosing acute HIV infections, before seroconversion, was low. There is an urgent need to develop and evaluate simple and affordable POC tests with high sensitivity and specificity for diagnosing individuals with acute HIV infection in resource-limited settings with high HIV prevalence.

HbA1c levels and circulating inflammatory proteins at onset of type 1 diabetes in children and adolescents.

Purpose: Type 1 diabetes is an autoimmune disease that often develops during childhood. Complications such as retinopathy often occur during the course of the disease. Studies to identify possible predictors of complications in type 1 diabetes are needed; in particular markers able to identify risk of complications long before they occur. The first aim of this study was to investigate plasma levels of sCD163, sST2 and Gal-3 at diagnosis of type 1 diabetes in children and adolescents. The second aim was to study their correlation to HbA1c in this study cohort. Methods: Patients (n = 242, 0-18 years) with type 1 diabetes, at Helsingborg's Hospital were included in this study and circulating levels of sCD163, sST2 and Gal-3 were investigated in plasma using commercially available DuoSet ELISA and supplementary ancillary kit. Results: Circulating sCD163 was significantly higher at diagnosis compared to after diagnosis (666 ± 318ng/ml and 505 ± 223ng/ml respectively; p < 0.001). Also sST2 was significantly higher (18.2 [12.7-25.6] ng/ml respectively 9.1 [6.3-13.5] ng/ml (p < 0.001), but Gal-3 levels did not differ from onset of diabetes to after diagnosis. HbA1c was shown to correlate to sCD163 (rs=0.36; p < 0.001), sST2 (rs=0.22; p = 0.016) and Gal-3 (rs=0.2; p = 0.020) in patients with a diabetes duration < 5 years. Conclusions: sCD163 levels increased in patients with recent-onset type 1 diabetes and the levels increased with higher HbA1c. Patients included in this study will be followed annually until the eventual development of diabetic complications, while continuously studying circulating levels of inflammatory proteins such as sCD163.

Vaccine-Induced Seroreactivity Impacts the Accuracy of HIV Testing Algorithms in Sub-Saharan Africa: An Exploratory Study.

The detection of vaccine-induced HIV antibody responses by rapid diagnostic tests (RDTs) may confound the interpretation of HIV testing results. We assessed the impact of vaccine-induced seroreactivity (VISR) on the diagnosis of HIV in sub-Saharan Africa. Samples collected from healthy participants of HIVIS and TaMoVac HIV vaccine trials after the final vaccination were analyzed for VISR using HIV testing algorithms used in Mozambique and Tanzania that employ two sequential RDTs. The samples were also tested for VISR using Enzygnost HIV Integral 4 ELISA and HIV western blot assays. Antibody titers to subtype C gp140 were determined using an in-house enzyme-linked immunosorbent assay (ELISA). The frequency of VISR was 93.4% (128/137) by Enzygnost HIV Integral 4 ELISA, and 66.4% (91/137) by western blot assay (WHO interpretation). The proportion of vaccine recipients that would have been misdiagnosed as HIV-positive in Mozambique was half of that in Tanzania: 26.3% (36/137) and 54.0% (74/137), respectively, p < 0.0001. In conclusion, the HIV RDTs and algorithms assessed here will potentially misclassify a large proportion of the HIV vaccine recipients if no other test is used. Increased efforts are needed to develop differential serological or molecular tools for use at the point of care.

The Prevalence, Incidence, and Risk Factors for HIV Among Female Sex Workers-A Cohort Being Prepared for a Phase IIb HIV Vaccine Trial in Dar es Salaam, Tanzania.

BACKGROUND: A cohort of female sex workers (FSWs) was established to determine HIV prevalence and incidence, and associated factors in preparation for a phase IIb HIV vaccine and pre-exposure prophylaxis trial (PrEPVacc). SETTING: A cohort of FSWs in Dar es Salaam, Tanzania. METHODS: FSWs aged 18-45 years were recruited using a respondent-driven sampling method. Social demographic data, HIV risk behavioral assessments, and blood samples for testing of HIV, syphilis, hepatitis B (HBV), and hepatitis C (HCV) infections were collected at baseline and then at 3, 6, 9, and 12 months. Poisson regressions were used to estimate the prevalence ratios for factors associated with HIV prevalence and to estimate the 12-month HIV incidence rate. RESULTS: Between October and December 2018, a total of 773 FSWs were screened for eligibility and 700 were enrolled. The baseline prevalence of HIV, syphilis, HBV, and HCV was 7.6%, 1.2%, 1.7%, and 1.0%, respectively. HIV prevalence was associated with older age, using illicit drugs, and being infected with syphilis, HBV, or HCV. Attendance at 12 months was 80% (562/700). Twenty-one FSWs seroconverted during follow-up, giving a 12-month HIV incidence rate of 3.45 per 100 person-years at risk (95% CI; 2.25-5.28/100 person-years at risk). The HIV incidence rate was higher among FSWs aged 18-24 years, FSWs who used drugs, and those diagnosed with syphilis, HBV, or HCV. CONCLUSION: The high HIV incidence rate and retention rate among FSWs enrolled into the cohort demonstrate that this population is suitable for participation in HIV prevention trials.

Duration of SARS-CoV-2 Immune Responses Up to Six Months Following Homologous or Heterologous Primary Immunization with ChAdOx1 nCoV-19 and BNT162b2 mRNA Vaccines.

Heterologous primary immunization against SARS-CoV-2 is part of applied recommendations. However, little is known about duration of immune responses after heterologous vaccine regimens. To evaluate duration of immune responses after primary vaccination with homologous adeno-vectored ChAdOx1 nCoV-19 vaccine (ChAd) or heterologous ChAd/BNT162b2 mRNA vaccine (BNT), anti-spike-IgG and SARS-CoV-2 VOC-neutralizing antibody responses were measured in 354 healthcare workers (HCW) at 2 weeks, 3 months, 5 months and 6 months after the second vaccine dose. T-cell responses were investigated using a whole blood interferon gamma (IFN-γ) release assay 2 weeks and 3 months post second vaccine dose. Two hundred and ten HCW immunized with homologous BNT were enrolled for comparison of antibody responses. In study participants naïve to SARS-CoV-2 prior to vaccination, heterologous ChAd/BNT resulted in 6-fold higher peak anti-spike IgG antibody titers compared to homologous ChAd vaccination. The half-life of antibody titers was 3.1 months (95% CI 2.8-3.6) following homologous ChAd vaccination and 1.9 months (95% CI 1.7-2.1) after heterologous vaccination, reducing the GMT difference between the groups to 3-fold 6 months post vaccination. Peak T-cell responses were stronger in ChAd/BNT vaccinees, but no significant difference was observed 3 months post vaccination. SARS-CoV-2 infection prior to vaccination resulted in substantially higher peak GMTs and IFN-γ levels and enhanced SARS-CoV-2 specific antibody and T cell responses over time. Heterologous primary SARS-CoV-2 immunization with ChAd and BNT elicits a stronger initial immune response compared to homologous vaccination with ChAd. However, although the differences in humoral responses remain over 6 months, the difference in SARS-CoV-2 specific T cell responses are no longer significant three months after vaccination.

Systematic comparison of HIV-1 Envelope-specific IgG responses induced by different vaccination regimens: Can we steer IgG recognition towards regions of viral vulnerability?

Immunogens and vaccination regimens can influence patterns of immune-epitope recognition, steering them towards or away from epitopes of potential viral vulnerability. HIV-1 envelope (Env)-specific antibodies targeting variable region 2 (V2) or 3 (V3) correlated with protection during the RV144 trial, however, it was suggested that the immunodominant V3 region might divert antibody responses away from other relevant sites. We mapped IgG responses against linear Env epitopes in five clinical HIV vaccine trials, revealing a specific pattern of Env targeting for each regimen. Notable V2 responses were only induced in trials administering CRF01_AE based immunogens, but targeting of V3 was seen in all trials, with the soluble, trimeric CN54gp140 protein eliciting robust V3 recognition. Strong V3 targeting was linked to greater overall response, increased number of total recognised antigenic regions, and where present, stronger V2 recognition. Hence, strong induction of V3-specific antibodies did not negatively impact the targeting of other linear epitopes in this study, suggesting that the induction of antibodies against V3 and other regions of potential viral vulnerability need not be necessarily mutually exclusive.

Transient hyperammonaemia following epileptic seizures in cats.

OBJECTIVES: The aim of this study was to determine whether transient postictal hyperammonaemia exists in cats. METHODS: The medical records of all feline patients that presented at a Swedish veterinary hospital between 2008 and 2018 were retrospectively reviewed to find those that had a recent or ongoing epileptic seizure. To qualify for inclusion, the medical record had to include information on at least one ammonia value taken in close proximity to, or during, an active seizure, the cat must have exceeded the normal upper limit of blood ammonia concentration on initial testing (reference interval 0-95 µmol/l), and there needed to be a follow-up ammonia value available within a maximum of 3 days. RESULTS: Five cats were included in the study, and they had blood ammonia concentrations on initial testing ranging from 146 to 195 µmol/l. They were all retested within a period of 2 h to 3 days of the original reading. All five cats had a spontaneous decrease in ammonia levels without any specific treatment for hyperammonaemia. CONCLUSIONS AND RELEVANCE: Pursuant to the findings of this retrospective study, transient hyperammonaemia may be noted after epileptic seizure in cats. Consequently, a differential diagnostic list in feline patients with hyperammonaemia could, depending on the context, include non-hepatic-related pathologies, such as epileptic seizures.

Evaluation of 11 SARS-CoV-2 antibody tests by using samples from patients with defined IgG antibody titers.

We evaluated the performance of 11 SARS-CoV-2 antibody tests using a reference set of heat-inactivated samples from 278 unexposed persons and 258 COVID-19 patients, some of whom contributed serial samples. The reference set included samples with a variation in SARS-CoV-2 IgG antibody titers, as determined by an in-house immunofluorescence assay (IFA). The five evaluated rapid diagnostic tests had a specificity of 99.0% and a sensitivity that ranged from 56.3 to 81.6% and decreased with low IFA IgG titers. The specificity was > 99% for five out of six platform-based tests, and when assessed using samples collected ≥ 22 days after symptom onset, two assays had a sensitivity of > 96%. These two assays also detected samples with low IFA titers more frequently than the other assays. In conclusion, the evaluated antibody tests showed a heterogeneity in their performances and only a few tests performed well with samples having low IFA IgG titers, an important aspect for diagnostics and epidemiological investigations.

Frequent and Durable Anti-HIV Envelope VIV2 IgG Responses Induced by HIV-1 DNA Priming and HIV-MVA Boosting in Healthy Tanzanian Volunteers.

We evaluated antibody responses to the human immunodeficiency virus (HIV) envelope variable regions 1 and 2 (V1V2) in 29 vaccinees who had received three HIV-1 DNA immunizations and two HIV-modified vaccinia virus Ankara (MVA) boosts in the phase I/II HIVIS03 vaccine trial. Twenty vaccinees received a third HIV-MVA boost after three years in the HIVIS06 trial. IgG and IgG antibody subclasses to gp70V1V2 proteins of HIV-1 A244, CN54, Consensus C, and Case A2 were analysed using an enzyme-linked immunosorbent assay (ELISA). Cyclic V2 peptides of A244, Consensus C, and MN were used in a surface plasmon resonance (SPR) assay. Four weeks after the second HIV-MVA, anti-V1V2 IgG antibodies to A244 were detected in 97% of HIVIS03 vaccinees, in 75% three years later, and in 95% after the third HIV-MVA. Anti-CN54 V1V2 IgG was detectable in 48% four weeks after the second HIV-MVA. The SPR data supported the findings. The IgG response was predominantly IgG1. Four weeks after the second HIV-MVA, 85% of vaccinees had IgG1 antibodies to V1V2 A244, which persisted in 25% for three-years. IgG3 and IgG4 antibodies to V1V2 A244 were rare. In conclusion, the HIV-DNA/MVA vaccine regimen induced durable V1V2 IgG antibody responses in a high proportion of vaccinees.

Induction of Identical IgG HIV-1 Envelope Epitope Recognition Patterns After Initial HIVIS-DNA/MVA-CMDR Immunization and a Late MVA-CMDR Boost.

In the RV144 trial, to date the only HIV-1 vaccine efficacy trial demonstrating a modestly reduced risk of HIV-1 acquisition, antibody responses toward the HIV Envelope protein (Env) variable (V) 2 and V3 regions were shown to be correlated with a reduced risk of infection. These potentially protective antibody responses, in parallel with the vaccine efficacy, however, waned quickly. Dissecting vaccine-induced IgG recognition of antigenic regions and their variants within the HIV-1 Env from different vaccine trials will aid in designing future HIV-1 immunogens and vaccination schedules. We, therefore, analyzed the IgG response toward linear HIV-1 Env epitopes elicited by a multi-clade, multigene HIVIS-DNA priming, and heterologous recombinant modified vaccinia virus Ankara (MVA-CMDR) boosting regimen (HIVIS03) and assessed whether a late MVA-CMDR boost 3 years after completion of the initial vaccination schedule (HIVIS06) restored antibody responses toward these epitopes. Here we report that vaccination schedule in the HIVIS03 trial elicited IgG responses against linear epitopes within the V2 and V3 tip as well as against the gp41 immunodominant region in a high proportion of vaccinees. Antibodies against the V2 and gp41 Env regions were restricted to variants with close homology to the MVA-CMDR immunogen sequence, while V3 responses were more cross-reactive. Boosting with a late third MVA-CMDR after 3 years effectively restored waned IgG responses to linear Env epitopes and induced targeting of identical antigenic regions and variants comparable to the previous combined HIVIS-DNA/MVA-CMDR regimen. Our findings support the notion that anti-HIV-1 Env responses, associated with a reduced risk of infection in RV144, could be maintained by regular boosting with a single dose of MVA-CMDR.

Frequent Anti-V1V2 Responses Induced by HIV-DNA Followed by HIV-MVA with or without CN54rgp140/GLA-AF in Healthy African Volunteers.

Antibody responses that correlated with reduced risk of HIV acquisition in the RV144 efficacy trial were assessed in healthy African volunteers who had been primed three times with HIV-DNA (subtype A, B, C) and then randomized into two groups; group 1 was boosted twice with HIV-MVA (CRF01_AE) and group 2 with the same HIV-MVA coadministered with subtype C envelope (Env) protein (CN54rgp140/GLA-AF). The fine specificity of plasma Env-specific antibody responses was mapped after the final vaccination using linear peptide microarray technology. Binding IgG antibodies to the V1V2 loop in CRF01_AE and subtype C Env and Env-specific IgA antibodies were determined using enzyme-linked immunosorbent assay. Functional antibody-dependent cellular cytotoxicity (ADCC)-mediating antibody responses were measured using luciferase assay. Mapping of linear epitopes within HIV-1 Env demonstrated strong targeting of the V1V2, V3, and the immunodominant region in gp41 in both groups, with additional recognition of two epitopes located in the C2 and C4 regions in group 2. A high frequency of V1V2-specific binding IgG antibody responses was detected to CRF01_AE (77%) and subtype C antigens (65%). In conclusion, coadministration of CN54rgp140/GLA-AF with HIV-MVA did not increase the frequency, breadth, or magnitude of anti-V1V2 responses or ADCC-mediating antibodies induced by boosting with HIV-MVA alone.