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Route of immunization can markedly influence the quality of immune response. Here, we show that intradermal (ID) but not intramuscular (IM) modified vaccinia Ankara (MVA) vaccinations provide protection from acquisition of intravaginal tier2 simian-human immunodeficiency virus (SHIV) challenges in female macaques. Both routes of vaccination induce comparable levels of serum IgG with neutralizing and non-neutralizing activities. The protection in MVA-ID group correlates positively with serum neutralizing and antibody-dependent phagocytic activities, and envelope-specific vaginal IgA; while the limited protection in MVA-IM group correlates only with serum neutralizing activity. MVA-ID immunizations induce greater germinal center Tfh and B cell responses, reduced the ratio of Th1 to Tfh cells in blood and showed lower activation of intermediate monocytes and inflammasome compared to MVA-IM immunizations. This lower innate activation correlates negatively with induction of Tfh responses. These data demonstrate that the MVA-ID vaccinations protect against intravaginal SHIV challenges by modulating the innate and T helper responses., (© 2023. Springer Nature Limited.)

Nitrosoalkanes (R-N═O; R = alkyl) are biological intermediates that form from the oxidative metabolism of various amine (RNH 2 ) drugs or from the reduction of nitroorganics (RNO 2 ). RNO compounds bind to and inhibit various heme proteins. However, structural information on the resulting Fe-RNO moieties remains limited. We report the preparation of ferrous wild-type and H64A sw Mb II -RNO derivatives (λ max 424 nm; R = Me, Et, Pr, i Pr) from the reactions of Mb III -H 2 O with dithionite and nitroalkanes. The apparent extent of formation of the wt Mb derivatives followed the order MeNO > EtNO > PrNO > i PrNO, whereas the order was the opposite for the H64A derivatives. Ferricyanide oxidation of the Mb II -RNO derivatives resulted in the formation of the ferric Mb III -H 2 O precursors with loss of the RNO ligands. X-ray crystal structures of the wt Mb II -RNO derivatives at 1.76-2.0 Å resoln. revealed N -binding of RNO to Fe and the presence of H-bonding interactions between the nitroso O-atoms and distal pocket His64. The nitroso O-atoms pointed in the general direction of the protein exterior, and the hydrophobic R groups pointed toward the protein interior. X-ray crystal structures for the H64A mutant derivatives were determined at 1.74-1.80 Å resoln. An analysis of the distal pocket amino acid surface landscape provided an explanation for the differences in ligand orientations adopted by the EtNO and PrNO ligands in their wt and H64A structures. Our results provide a good baseline for the structural analysis of RNO binding to heme proteins possessing small distal pockets.

The gut microbiota has a fundamental role in the development and the maturation of the host immune system. Both innate and adaptive immune cells have critical functions in microbial pathogen containment and clearance, but the regulation of the commensal microbiome ecosystem in the gastrointestinal tract by these major immune cell populations is incompletely defined. The role of specific innate and adaptive immune cell in the regulation of the microbiota in the intestinal tract biogeographically was investigated. Dendritic cells, macrophages, CD4+ T-cells, CD8+ T-cells, and B-cells were depleted using monoclonal antibodies and clodronate liposomes, and the microbial communities were determined by 16S rRNA gene sequencing. With specific immune cell depletion, distinct microbiota changes were observed. In general, immune cell depleted mice had higher microbiota richness and evenness at all gut anatomical sites. At each gut segment, samples from immune cell-depleted animals clustered away from the isotype/liposome control mice. This was especially dramatic for the small intestinal microbiota. Specifically, Enterobacteriaceae, Bacteroides acidifaciens, and Mucispirillum schaedleri were highly enriched in the mucosa and lumen of the small intestine in immune cell-deficient animals. Further, the mucosal microbiota had higher microbiota evenness compared with luminal microbiota at all gut segments, and the UniFrac distance between B cell depleted and isotype control mice was the largest in the duodenum followed by the ileum and colon. Taken together, the data suggest that innate and adaptive immune cells specifically contribute to the regulation of the gut microbiota's biogeographical distribution along the gastrointestinal tract, and microbiota in the duodenum mucosa are more responsive to host immune changes compared with other anatomical sites., (© 2022 The Societies and John Wiley & Sons Australia, Ltd.)

Stabilized HIV-1 envelope (Env) trimers elicit tier 2 autologous neutralizing antibody (nAb) responses in immunized animals. We previously demonstrated that BG505 SOSIP.664.T332N gp140 (BG505 SOSIP) immunization of rhesus macaques (RM) provided robust protection against autologous intra-vaginal simian-human immunodeficiency virus (SHIV) challenge that was predicted by high serum nAb titers. Here, we show that nAb in these protected RM targeted a glycan hole proximal to residue 465 in gp120 in all cases. nAb also targeted another glycan hole at residues 241/289 and an epitope in V1 at varying frequencies. Non-neutralizing antibodies directed at N611-shielded epitopes in gp41 were also present but were more prevalent in RM with low nAb titers. Longitudinal analysis demonstrated that nAb broadened in some RM during sequential immunization but remained focused in others, the latter being associated with increases in nAb titer. Thirty-eight monoclonal antibodies (mAbs) isolated from a protected RM with an exceptionally high serum neutralization titer bound to the trimer in ELISA, and four of the mAbs potently neutralized the BG505 Env pseudovirus (PV) and SHIV. The four neutralizing mAbs were clonally related and targeted the 465 glycan hole to varying degrees, mimicking the serum. The data demonstrate that the C3/465 glycan hole cluster was the dominant neutralization target in high titer protected RM, despite other co-circulating neutralizing and non-neutralizing specificities. The isolation of a neutralizing mAb family argues that clonotype expansion occurred during BG505 SOSIP immunization, leading to high titer, protective nAb and setting a desirable benchmark for HIV vaccines., Competing Interests: The authors have declared that no competing interests exist.

Recent efforts toward an HIV vaccine focus on inducing broadly neutralizing antibodies, but eliciting both neutralizing antibodies (nAbs) and cellular responses may be superior. Here, we immunized macaques with an HIV envelope trimer, either alone to induce nAbs, or together with a heterologous viral vector regimen to elicit nAbs and cellular immunity, including CD8 + tissue-resident memory T cells. After ten vaginal challenges with autologous virus, protection was observed in both vaccine groups at 53.3% and 66.7%, respectively. A nAb titer >300 was generally associated with protection but in the heterologous viral vector + nAb group, titers <300 were sufficient. In this group, protection was durable as the animals resisted six more challenges 5 months later. Antigen stimulation of T cells in ex vivo vaginal tissue cultures triggered antiviral responses in myeloid and CD4 + T cells. We propose that cellular immune responses reduce the threshold of nAbs required to confer superior and durable protection.

With almost 2 million new HIV-1 infections in 2018, a highly effective vaccine is imperative. Vaccine-elicited HIV-1 antibodies contribute to protection through multiple nonneutralizing activities, but the exact mechanisms remain unknown. In this issue of the JCI, Neidich and associates sought to determine how antibodies contributed to reducing the risk of HIV-1 acquisition in a phase IIb preventative vaccine efficacy trial, HVTN 505. Their studies revealed that antibody-dependent cellular phagocytosis (ADCP) and FcγRIIa binding were strongly associated with reduced HIV-1 risk; however, HIV-1 envelope-specific IgG3, IgA; and host FcγRIIa genotype also influenced risk. This study highlights the intricate interactions between antibodies and innate immune functions in humans.

The goals of preclinical HIV vaccine studies in nonhuman primates are to develop and test different approaches for their ability to generate protective immunity. Here, we compared the impact of 7 different vaccine modalities, all expressing the HIV-1 1086.C clade C envelope (Env), on (i) the magnitude and durability of antigen-specific serum antibody responses and (ii) autologous and heterologous neutralizing antibody capacity. These vaccination regimens included immunization with different combinations of DNA, modified vaccinia virus Ankara (MVA), soluble gp140 protein, and different adjuvants. Serum samples collected from 130 immunized monkeys at two key time points were analyzed using the TZM-bl cell assay: at 2 weeks after the final immunization (week 40/41) and on the day of challenge (week 58). Key initial findings were that inclusion of a gp140 protein boost had a significant impact on the magnitude and durability of Env-specific IgG antibodies, and addition of 3M-052 adjuvant was associated with better neutralizing activity against the SHIV1157ipd3N4 challenge virus and a heterologous HIV-1 CRF01 Env, CNE8. We measured neutralization against a panel of 12 tier 2 Envs using a newly described computational tool to quantify serum neutralization potency by factoring in the predetermined neutralization tier of each reference Env. This analysis revealed modest neutralization breadth, with DNA/MVA immunization followed by gp140 protein boosts in 3M-052 adjuvant producing the best scores. This study highlights that protein-containing regimens provide a solid foundation for the further development of novel adjuvants and inclusion of trimeric Env immunogens that could eventually elicit a higher level of neutralizing antibody breadth. IMPORTANCE Despite much progress, we still do not have a clear understanding of how to elicit a protective neutralizing antibody response against HIV-1 through vaccination. There have been great strides in the development of envelope immunogens that mimic the virus particle, but less is known about how different vaccination modalities and adjuvants contribute to shaping the antibody response. We compared seven different vaccines that were administered to rhesus macaques and that delivered the same envelope protein through various modalities and with different adjuvants. The results demonstrate that some vaccine components are better than others at eliciting neutralizing antibodies with breadth., (Copyright © 2019 American Society for Microbiology.)

Background: Pneumocystis pneumonia is a major cause of morbidity and mortality in patients infected with HIV/AIDS. In this study, we evaluated the intestinal microbial communities associated with the development of experimental Pneumocystis pneumonia, as there is growing evidence that the intestinal microbiota is critical for host defense against fungal pathogens., Methods: C57BL/6 mice were infected with live Pneumocystis murina (P. murina) via intratracheal inoculation and sacrificed 7 and 14 days postinfection for microbiota analysis. In addition, we evaluated the intestinal microbiota from CD4+ T cell depleted mice infected with P. murina., Results: We found that the diversity of the intestinal microbial community was significantly altered by respiratory infection with P. murina. Specifically, mice infected with P. murina had altered microbial populations, as judged by changes in diversity metrics and relative taxa abundances. We also found that CD4+ T cell depleted mice infected with P. murina exhibited significantly altered intestinal microbiota that was distinct from immunocompetent mice infected with P. murina, suggesting that loss of CD4+ T cells may also affects the intestinal microbiota in the setting of Pneumocystis pneumonia. Finally, we employed a predictive metagenomics approach to evaluate various microbial features. We found that Pneumocystis pneumonia significantly alters the intestinal microbiota's inferred functional potential for carbohydrate, energy, and xenobiotic metabolism, as well as signal transduction pathways., Conclusions: Our study provides insight into specific-microbial clades and inferred microbial functional pathways associated with Pneumocystis pneumonia. Our data also suggest a role for the gut-lung axis in host defense in the lung., Competing Interests: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.

Pneumocystis pneumonia is a major cause of morbidity and mortality among immunocompromised patients, especially in the context of HIV/AIDS. In the murine model of Pneumocystis pneumonia, CD4(+) T-cells are required for clearance of a primary infection of Pneumocystis, but not the memory recall response. We hypothesized that the memory recall response in the absence of CD4(+) T-cells is mediated by a robust memory humoral response, CD8(+) T-cells, and IgG-mediated phagocytosis by alveolar macrophages. To investigate the role of CD8(+) T-cells and alveolar macrophages in the immune memory response to Pneumocystis, mice previously challenged with Pneumocystis were depleted of CD8(+) T-cells or alveolar macrophages prior to re-infection. Mice depleted of CD4(+) T-cells prior to secondary challenge cleared Pneumocystis infection within 48 h identical to immunocompetent mice during a secondary memory recall response. However, loss of CD8(+) T-cells or macrophages prior to the memory recall response significantly impaired Pneumocystis clearance. Specifically, mice depleted of CD8(+) T-cells or alveolar macrophages had significantly higher fungal burden in the lungs. Furthermore, loss of alveolar macrophages significantly skewed the lung CD8(+) T-cell response toward a terminally differentiated effector memory population and increased the percentage of IFN-γ(+) CD8(+) T-cells. Finally, Pneumocystis-infected animals produced significantly more bone marrow plasma cells and Pneumocystis-specific IgG significantly increased macrophage-mediated killing of Pneumocystis in vitro. These data suggest that secondary immune memory responses to Pneumocystis are mediated, in part, by CD8(+) T-cells, alveolar macrophages, and the production of Pneumocystis-specific IgG.

Immunosuppression associated with human immunodeficiency virus (HIV) infection impacts all components of host defense against pulmonary infection. Cells within the lung have altered immune function and are important reservoirs for HIV infection. The host immune response to infected lung cells further compromises responses to a secondary pathogenic insult. In the upper respiratory tract, mucociliary function is impaired and there are decreased levels of salivary immunoglobulin A. Host defenses in the lower respiratory tract are controlled by alveolar macrophages, lymphocytes, and polymorphonuclear leukocytes. As HIV infection progresses, lung CD4 T cells are reduced in number causing a lack of activation signals from CD4 T cells and impaired defense by macrophages. CD8 T cells, on the other hand, are increased in number and cause lymphocytic alveolitis. Specific antibody responses by B-lymphocytes are decreased and opsonization of microorganisms is impaired. These observed defects in host defense of the respiratory tract explain the susceptibility of HIV-infected persons for oropharyngeal candidiasis, bacterial pneumonia, Pneumocystis pneumonia, and other opportunistic infections., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Pneumocystis pneumonia is a major cause of morbidity and mortality in immunocompromised patients, particularly those infected with HIV. In this study, we evaluated the potential of oral immunization with live Pneumocystis to elicit protection against respiratory infection with Pneumocystis murina. C57BL/6 mice vaccinated with live P. murina using a prime-boost vaccination strategy were protected from a subsequent lung challenge with P. murina at 2, 7, 14, and 28 d postinfection even after CD4(+) T cell depletion. Specifically, vaccinated immunocompetent mice had significantly faster clearance than unvaccinated immunocompetent mice and unvaccinated CD4-depleted mice remained persistently infected with P. murina. Vaccination also increased numbers of CD4(+) T cells, CD8(+) T cells, CD19(+) B cells, and CD11b(+) macrophages in the lungs following respiratory infection. In addition, levels of lung, serum, and fecal P. murina-specific IgG and IgA were increased in vaccinated animals. Furthermore, administration of serum from vaccinated mice significantly reduced Pneumocystis lung burden in infected animals compared with control serum. We also found that the diversity of the intestinal microbial community was altered by oral immunization with P. murina. To our knowledge, our data demonstrate for the first time that an oral vaccination strategy prevents Pneumocystis infection., (Copyright © 2016 by The American Association of Immunologists, Inc.)

A survey on the nematode control strategies utilized by dairy farmers in Southeastern Brazil, a region accounting for 46.4% of the national milk production, was conducted through interviews with farmers. To select the producers to be interviewed the region was split into 16 non-contiguous clusters, according to the level of milk production. A systematic sample was then selected in each of the clusters. The interview questionnaire consisted of one-way, multiple-choice and open-ended questions. Data collected were represented by numbers and digitized on a data base (Epi Info, version 5.01b) and analyzed. Out of the 89 farmers interviewed, 37.5% deworm their herd after clinical signs and 62.5% preventively. Generally, anthelmintics are applied from one to 12 times a year (average of 3.79 times a year) in all age categories of animals. Of the anthelmintics used in the last deworming, imidazothiazole was used exclusively by 17.1% of the farmers, benzimidazole by 9.8% and avermectin by 18.3%, while 55.8% used more than one anthelmintic class to deworm their animals. To choose the dosage, most farmers consult the product label (94.8%) and determine the volume to be applied, based on an estimate of the average body weight of each animal (62.9%). Improvements in the general appearance of the herd and weight gains of growing animals were observed by most farmers after deworming (87.3%). However, most of them (66.2%) recalled interrupting the use of some compound in the last few years, due to the detection of no improvement following treatment (32.7%), rising costs of the medication (28.6%), adverse reaction (8.2%), product not available at the time of purchase (4.1%) and decision to change the compound in use (10.2%). Most farmers (95.3%) intend to continue using the same control measures in the following year. Veterinarians play an important role in the farmer's choice to deworm their animals, as many seek advice from them. Therefore, programs aimed at technology transfer should include continuous updates on the subject, especially for veterinarians.

Harposporium anguillulae is a common nematophagous fungus which colonizes cattle pats deposited in Brachiaria decumbens pasture in Brazil. To assess its possible effect on free living stages of trichostrongylid nematodes, the reduction of Haemonchus contortus infective stages in sheep faecal culture was evaluated in vitro. Addition of 300,000 conidia per gram of faeces caused a reduction of 99.5% compared to the control group. This result indicates that H. anguillulae could be a candidate for the development of strategies for the biological control of trichostrongylid nematodes.

Two worm-free calves were allowed to graze on irrigated pasture with a naturally infected herd for each of 34 one-month periods from November 1979 to August 1982. After each grazing period, the calves were transferred to a cement-floored pen for 3 weeks and then were euthanatized and necropsied. Ostertagia ostertagi and Cooperia oncophora were the most prevalent species of nematodes recovered. Adults and larvae of Ostertagia spp and Cooperia spp were most numerous in winter and spring and least numerous during summer. The proportions of Ostertagia spp that were inhibited as fourth-stage larvae increased in late fall, peaked from March through April, and then decreased to low values during summer. The maximal inhibition in 1980, 1981, and 1982 was 72, 65, and 62%, respectively. The number of larval Cooperia spp was highest in winter months and, except for one grazing period when 55% of the Cooperia spp were larvae, the total numbers represented less than 15% of the nematode population during all grazing periods. Other nematodes encountered were Trichostrongylus axei, Haemonchus spp, O lyrata, and O occidentalis in the abomasum; C surnabata, C punctata, Nematodirus helvetianus, T colubriformis, and Bunostomum phlebotomum in the small intestine; and Oesophagostomum venulosum and Trichuris ovis in the large intestine.

Groups of 10 goats, harbouring both naturally acquired and experimental infections of gastrointestinal nematodes, were drenched with either levamisole (5 mg kg-1), albendazole (3.8 mg kg-1) or parbendazole (15 mg kg-1), or remained untreated. Haemonchus contortus was the numerically dominant infection, with Strongyloides papillosus, Trichostrongylus colubriformis and Oesophagostomum columbianum also present. At 5-6 days post-treatment, goats were killed and necropsied. Post-mortem worm counts showed that the reduction in mean total worm burdens was 57.4% in levamisole-treated animals, 71.1% in the albendazole group and 85.1% in the parbendazole group. Reductions for H. contortus were 80.2, 87.9 and 83.9% in the levamisole-, albendazole- and parbendazole-treated groups, respectively. These data indicate that the anthelmintics in question are not being applied at an adequate dose rate for goats, and/or resistance to anthelmintics is occurring in the field in Pernambuco State, northeast Brazil.

Eighteen female Holstein calves, raised as natural herd additions under conditions typical of a well-managed midwestern United States dairy farm, were used in a natural-exposure study to determine the anticoccidial efficacies of lasalocid and decoquinate. Calves were allotted to 6 treatment blocks of 3 calves each as they were weaned. Within each block, calves were randomly assigned to be given either lasalocid or decoquinate or to remain as a nonmedicated control. Calves were given medication for 90 days and remained separated from other calves for 120 days. Adjusted weight gains were consistently greater in calves that were given medication; however, differences were not statistically significant. Fecal specimens were obtained from calves at weekly intervals during the study. Overall, oocyst shedding was low. During the medication period, quantitative mean fecal shedding of oocysts was reduced eightfold in calves given decoquinate and four-fold in calves given lasalocid, as compared with nonmedicated control calves. During the period following the medication period, calves that had been controls shed fewer oocysts than did calves that had previously been given medication. A pairwise comparison of the proportion of specimens that were oocyst-positive was made to assess qualitative oocyst shedding among treatment groups. During the medication period, qualitative oocyst shedding (all species, Eimeria bovis, E zuernii, species other than E bovis and E zuernii) was greater in controls than in either lasalocid-or decoquinate-treated groups. Like-wise, lasalocid-medicated calves shed oocysts more frequently than did the decoquinate-medicated group. After medication, qualitative findings were reversed. Little diarrhea was noticed in treatment or control calves during the study.(ABSTRACT TRUNCATED AT 250 WORDS)

During each of 36 1-month periods from April 1979 to March 1982, 3-4 goats selected from typical farms were necropsied and examined for gastrointestinal nematodes. The goats were male, 12 months old, born on the farm and raised without any anthelmintic application. At the beginning of each month, from April 1981 to March 1982, three 12-month-old male goats shown to be free of gastrointestinal nematodes after anthelmintic treatment were grazed with a flock of naturally infected goats (tracer goats). At the end of each month, these goats were placed on a cement-floored pen and were maintained there for 4 weeks prior to necropsy and examination for gastrointestinal nematodes. Every goat examined was found to be parasitized by more than one species of nematode. Haemonchus contortus, Strongyloides papillosus and Oesophagostomum columbianum were the most prevalent nematodes found. Total worm burdens present in the farm animals were highest during late rainy/early dry season (March-June) and lowest in mid-rainy season (January-February). The acquisition of nematodes by tracer goats occurred mainly from mid-rainy to early dry season (January-June).