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Objectives Regulatory T cells (Tregs) are widely recognised as a subset of CD4+CD25+FOXP3+ T cells that have a key role in maintaining immune homeostasis. The impact of HIV‐1 infection on immunological properties and effector functions of Tregs has remained the topic of debate and controversy. In the present study, we investigated transcriptional profile and functional properties of Tregs in HIV‐1‐infected individuals either receiving antiretroviral therapy (ART, n = 50) or long‐term non‐progressors (LTNPs, n = 24) compared to healthy controls (HCs, n = 38). Methods RNA sequencing (RNAseq), flow cytometry‐based immunophenotyping and functional assays were performed to study Tregs in different HIV cohorts. Results Our RNAseq analysis revealed that Tregs exhibit different transcriptional profiles in HIV‐infected individuals. While Tregs from patients on ART upregulate pathways associated with a more suppressive (activated) phenotype, Tregs in LTNPs exhibit upregulation of pathways associated with impaired suppressive properties. These observations may explain a higher propensity for autoimmune diseases in LTNPs. Also, we found substantial upregulation of HLA‐F mRNA and HLA‐F protein in Tregs from HIV‐infected subjects compared to healthy individuals. These observations highlight a potential role for this non‐classical HLA in Tregs in the context of HIV infection, which should be investigated further in other chronic viral infections and cancer. Conclusion Our study has provided a novel insight into Tregs at the transcriptional and functional levels in different HIV‐infected groups., This study provides a novel insight into regulatory T cells (Tregs) at the transcriptional and functional levels in HIV‐infected individuals on antiretroviral therapy versus long‐term non‐progressors (LTNPs). A lack of immunosuppressive properties of Tregs in LTNPs may explain the higher propensity for autoimmune diseases in these individuals. ​

Background In the absence of antiretroviral treatments (ARTs), a small group of individuals infected with HIV, including long-term non-progressors (LTNPs) who maintain high levels of CD4+ T cells for more than 7–10 years in the absence of ART and in particular a subgroup of LTNPs, elite controllers (ECs), who have low levels of viremia, remain clinically and/or immunologically stable for years. However, the mechanism of stable disease progression in LTNPs and ECs needs to be elucidated to help those infected with HIV-1 remain healthy. In this study, to identify the characteristics of gene expression profiles and biomarkers in LTNPs, we performed a meta-analysis using multiple gene expression profiles among LTNPs, individuals infected with HIV-1 without ART, individuals infected with HIV-1 with ART, and healthy controls. Methods The gene expression profiles obtained from the Gene Expression Omnibus (GEO) microarray data repositories were classified into three groups: LTNPs versus healthy controls (first group, 3 studies), LTNPs versus patients infected with HIV-1 without ART (second group, 3 studies), and LTNPs versus patients infected with HIV-1 with ART (third group, 3 studies). In addition, we considered a fourth group, patients infected with HIV-1 without ART versus healthy controls (3 studies), to exclude genes associated with HIV-1 infection in the three groups. For each group, we performed a meta-analysis using the RankProd method to identify and compare the differentially expressed genes (DEGs) in the three groups. Results We identified the 14 common DEGs in the three groups when comparing them with each other. Most belonged to immune responses, antigen processing and presentation, the interferon-gamma-mediated signaling pathway, and T cell co-stimulation. Of these DEGs, PHLDA1 was up-regulated and ACTB and ACTG1 were down-regulated in all three groups. However, the rest of the up- or down-regulated genes were discordant in the three groups. Additionally, ACTB and ACTG1 are known to inhibit viral assembly and production, and THBS1 is known to inhibit HIV-1 infection. Conclusions These results suggest that significant genes identified in a meta-analysis provide clues to the cause of delayed disease progression and give a deeper understanding of HIV pathogenesis in LTNPs. Electronic supplementary material The online version of this article (10.1186/s12920-018-0443-x) contains supplementary material, which is available to authorized users.

Viremic non-progressors (VNPs), a distinct group of HIV-1-infected individuals, exhibit no signs of disease progression and maintain persistently elevated CD4+ T cell counts for several years despite high viral replication. Comprehensive characterization of homeostatic cellular immune signatures in VNPs can provide unique insights into mechanisms responsible for coping with viral pathogenesis as well as identifying strategies for immune restoration under clinically relevant settings such as antiretroviral therapy (ART) failure. We report a novel homeostatic signature in VNPs, the preservation of the central memory CD4+ T cell (CD4+ T CM ) compartment. In addition, CD4+ TCM preservation was supported by ongoing interleukin-7 (IL-7)-mediated thymic repopulation of naive CD4+ T cells leading to intact CD4+ T cell homeostasis in VNPs. Regulatory T cell (Treg) expansion was found to be a function of preserved CD4+ T cell count and CD4+ T cell activation independent of disease status. However, in light of continual depletion of CD4+ T cell count in progressors but not in VNPs, Tregs appear to be involved in lack of disease progression despite high viremia. In addition to these homeostatic mechanisms resisting CD4+ T cell depletion in VNPs, a relative diminution of terminally differentiated effector subset was observed exclusively in these individuals that might ameliorate consequences of high viral replication. VNPs also shared signatures of impaired CD8+ T cell cytotoxic function with progressors evidenced by increased exhaustion (PD-1 upregulation) and CD127 (IL-7Rα) downregulation contributing to persistent viremia. Thus, the homeostatic immune signatures reported in our study suggest a complex multifactorial mechanism accounting for non-progression in VNPs.

Background Disease progression in the absence of therapy varies significantly in mono-HIV and HCV infected individuals. Virus-specific CD8+ T cells play an important role in restricting lentiviral replication and determining the rate of disease progression during HIV and HCV mono- and co-infection. Thus, understanding the similarities in the characteristics of CD8+ T cells in mono-HIV and HCV infection at the transcriptomic level contributes to the development of antiviral therapy. In this study, a meta-analysis of CD8+ T cell gene expression profiles derived from mono-HIV and HCV infected individuals at different stages of disease progression, was conducted to understand the common changes experienced by CD8+ T cells. Methods Five microarray datasets, reporting CD8+ T cell mRNA expression of the mono-HIV and HCV infected patients, were retrieved from Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) were identified via integrative meta-analysis of expression data (INMEX) program. Network analysis methods were used to assess protein–protein interaction (PPI) networks, Gene Ontology (GO) terms and pathway enrichment for DEGs. MirDIP and miRDB online prediction tools were used to predict potential microRNAs (miRNAs) targeting hub genes. Results First, we identified 625 and 154 DEGs in the CD8+ T cells originating from mono-HIV and HCV chronic progressor patients, respectively, compared to healthy individuals. Among them, interferon-stimulated genes (ISGs) including ISG15, IFIT3, ILI44L, CXCL8, FPR1 and TLR2, were upregulated after mono-HIV and HCV infection. Pathway enrichment analysis of DEGs showed that the “cytokine–cytokine receptor interaction” and “NF-kappa B” signaling pathways were upregulated after mono-HIV and HCV infection. In addition, we identified 92 and 50 DEGs in the CD8+ T cells of HIV non-progressor and HCV resolver patients, respectively, compared with corresponding chronic progressors. We observed attenuated mitosis and reduced ISG expression in HIV non-progressors and HCV resolvers compared with the corresponding chronic progressors. Finally, we identified miRNA-143-3p, predicted to target both IFIT3 in HIV and STAT5A in HCV infection. Conclusions We identified DEGs and transcriptional patterns in mono-HIV and HCV infected individuals at different stages of disease progression and identified miRNA-143-3p with potential to intervene disease progression, which provides a new strategy for developing targeted therapies.

Factor de impacto: 4,242 Q1 Long-term non-progressors (LTNP) and elite controllers (EC) represent spontaneous natural models of efficient HIV-1 response in the absence of treatment. The main purposes of this work are to describe the miRNome of HIV-1 infected patients with different extreme phenotypes and identify potentially altered pathways regulated by differentially expressed (DE) miRNAs. The miRNomes from peripheral blood mononuclear cells (PBMCs) of dual phenotype EC-LTNP or LTNP with detectable viremia and HIV-infected patients with typical progression before and after treatment, were obtained through miRNA-Seq and compared among them. The administration of treatment produces 18 DE miRNAs in typical progressors. LTNP condition shows 14 DE miRNA when compared to typical progressors, allowing LTNP phenotype differentiation. A set of four miRNAs: miR-144-3p, miR-18a-5p, miR-451a, and miR-324 is strongly downregulated in LTNP and related to protein regulation as AKT, mTOR, ERK or IKK, involved in immune response pathways. Deregulation of 28 miRNA is observed between EC-LTNP and viremic-LTNP, including previously described anti-HIV miRNAs: miR-29a, associated with LTNP phenotype, and miR-155, targeting different pre-integration complexes such as ADAM10 and TNPO3. A holistic perspective of the changes observed in the miRNome of patients with different phenotypes of HIV-control and non-progression is provided. This study has been conducted within the Spanish AIDS Research Network (RIS), funded by Instituto de Salud Carlos III (Plan Estatal de I+D+I 2013-2016) and co-funded by European Regional Development Fund (ERDF) “A way to build Europe” (RD12/0017/0015 and RD16CIII/0002/0001 projects), and the European Union’s Horizon 2020 Research and Innovation Programme under grant number 681137. The HIV BioBank, integrated in the Spanish AIDS Research Network, is partially funded by the RD16/0025/0019 project as part of the Plan Nacional R + D + I and cofinanced by ISCIII- Subdirección General de Evaluación and el Fondo Europeo de Desarrollo Regional (FEDER). R.A.-S. was supported by the Ministry of Innovation, Science and Universities predoctoral funding (FPU18/05527) and H.E.T.-T. by the ISCIII-PFIS predoctoral program (FI14CIII/00014). Sí

Human immunodeficiency virus (HIV)-infected long-term non-progressors (LTNPs) are of particular importance because of their unique disease progression characteristics. Defined by the maintenance of normal CD4+T cells after more than 8 years of infection, these LTNPs are heterogeneous. Some LTNPs exhibit ongoing viral production, while others do not and are able to control viral production. The underlying basis for this heterogeneity has not been clearly elucidated. In this study, the miRNA expression profiles of LTNPs were assessed. The levels of microRNA-19b (miR-19b) were found to be significantly increased in peripheral blood mononuclear cells of LTNPs with lower rather than higher viral load. We made clear that miR-19b may regulate CD8+T cell functions in HIV infection, which has not been addressed before. Overexpression of miR-19b promoted CD8+T cell proliferation, as well as interferon-γ and granzyme B expression, while inhibiting CD8+T cells apoptosis induced by anti-CD3/CD28 stimulation. The target of miR-19b was found to be the "phosphatase and tensin homolog", which regulates CD8+T cells function during HIV infections. Furthermore, we found that miR-19b can directly inhibit viral production in in-vitro HIV infected T cells. These results highlight the importance of miR-19b to control viral levels, which facilitate an understanding of human immunodeficiency virus pathogenesis and provide potential targets for improved immune intervention.

Human immunodeficiency virus (HIV)-infected long-term non-progressors (LTNPs) are of particular importance because of their unique disease progression characteristics. Defined by the maintenance of normal CD4+T cells after more than 8 years of infection, these LTNPs are heterogeneous. Some LTNPs exhibit ongoing viral production, while others do not and are able to control viral production. The underlying basis for this heterogeneity has not been clearly elucidated. In this study, the miRNA expression profiles of LTNPs were assessed. The levels of microRNA-19b (miR-19b) were found to be significantly increased in peripheral blood mononuclear cells of LTNPs with lower rather than higher viral load. We made clear that miR-19b may regulate CD8+T cell functions in HIV infection, which has not been addressed before. Overexpression of miR-19b promoted CD8+T cell proliferation, as well as interferon-γ and granzyme B expression, while inhibiting CD8+T cells apoptosis induced by anti-CD3/CD28 stimulation. The target of miR-19b was found to be the “phosphatase and tensin homolog”, which regulates CD8+T cells function during HIV infections. Furthermore, we found that miR-19b can directly inhibit viral production in in-vitro HIV infected T cells. These results highlight the importance of miR-19b to control viral levels, which facilitate an understanding of human immunodeficiency virus pathogenesis and provide potential targets for improved immune intervention.

Several B cell defects are reported in HIV-1 infected individuals including variation in B cell subsets, polyclonal B cell activation and exhaustion, with broadly neutralizing antibodies elicited in less than 10–20% of the infected population. HIV-1 disease progression is faster in children than adults. B Lymphocyte Stimulator (BLyS), expressed on dendritic cells (DCs), is a key regulator of B cell homeostasis. Understanding how DCs influence B cell phenotype and functionality (viral neutralization), thereby HIV-1 disease outcome in infected children, is important to develop interventional strategies for restoration of B cell function. In this study, a total of 38 vertically transmitted HIV-1 infected antiretroviral therapy (ART) naïve children and 25 seronegative controls were recruited. Based on the CD4 counts and years post-infection, infected children were categorized as long-term non-progressors (LTNPs) (n = 20) and progressors (n = 18). Eight of these progressors were followed up at 6–12 months post-ART. Percentages (%) of DCs, B cell subsets, and expression of BLyS on DCs were analyzed by flow-cytometry. Plasma levels of B cell growth factors were measured by ELISA and viral neutralization activity was determined using TZM-bl assay. Lower (%) of myeloid DCs (mDCs), plasmacytoid DCs, and high expression of BLyS on mDCs were observed in HIV-1 infected progressors than seronegative controls. Progressors showed lower % of naive B cells, resting memory B cells and higher % of mature activated, tissue-like memory B cells as compared to seronegative controls. Higher plasma levels of IL-4, IL-6, IL-10, and IgA were observed in progressors vs. seronegative controls. Plasma levels of IgG were high in progressors and in LTNPs than seronegative controls, suggesting persistence of hypergammaglobulinemia at all stages of disease. High plasma levels of BLyS in progressors positively correlated with poor viral neutralizing activity. Interestingly on follow up, treatment naïve progressors, post-ART showed increase in resting memory B cells along with reduction in plasma BLyS levels that correlated with improvement in viral neutralization. This is the first study to demonstrate that reduction in plasma BLyS levels correlates with restoration of B cell function, in terms of viral neutralization in HIV-1-infected children.

Despite significant contributions of monocytes to HIV persistence, the genomic basis of HIV-infection of monocytes and its association with plasma viremia remain elusive. To understand HIV interactions with monocytes during disease progression, monocytic transcriptomes from long-term non-progressors (LTNP), HIV+ patients with viral load 1000, and seronegative controls were analyzed using Illumina microarray. Differentially expressed genes were identified (fold change >2; adjusted p

Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. DOI: http://dx.doi.org/10.7554/eLife.09115.001, eLife digest Many people with HIV infections are able to live relatively normal lives thanks to major advances in drug therapies. A cure, however, remains elusive. One reason for this is that the virus can hide in certain types of human cells, where it is protected from the immune system and the effects of “antiretroviral” drugs. This creates reservoirs of virus particles in the body that can quickly multiply and spread if treatment stops. Some people who become infected with HIV are able to contain the virus without the help of drug treatments. These individuals – known as long-term non-progressors – do not become ill and only have low numbers of HIV particles in reservoirs. People who receive treatment early in the course of an HIV infection also have fewer viruses in reservoirs and are less likely to develop severe illness. Therefore, it might be possible to develop a “functional” cure that may not completely eliminate the virus from the body, but would prevent illness and allow the individuals to eventually stop taking antiretroviral drugs. Now, Malatinkova, De Spiegelaere et al. studied samples from 84 patients with HIV-1 to find how much effect an early start to treatment has on the amount of the virus in reservoirs. People who started treatment soon after infection had lower levels of HIV-1 in their blood than people who started treatment later (even after 10 years of treatment). However, patients that started treatment early had higher levels of HIV-1 in the blood than the patients who were long-term non-progressors. All the patients had similar levels of HIV-1 in tissue samples taken from the rectum, regardless of when they started treatment. The experiments suggest that HIV-1 reservoirs form very soon after infection. Malatinkova, De Spiegelaere et al. found that in addition to reducing reservoirs of HIV-1, an early start to drug treatment reduced the ability of the virus to make copies of its genetic code. People who started treatment earlier also had healthier immune cells. Together, the experiments support the benefits of starting drug treatments as soon as possible after a person is infected with HIV-1. It is important to further characterize thoroughly the viral reservoir in patients with limited HIV-1 reservoirs and to look for other immune factors involved in virus control, in the search for a functional cure of HIV. DOI: http://dx.doi.org/10.7554/eLife.09115.002

The field of HIV prevention has indeed progressed in leaps and bounds, but with major limitations of the current prevention and treatment options, the world remains desperate for an HIV vaccine. Sadly, this continues to be elusive as more than 30 years since its discovery there is no licensed HIV vaccine. Research aiming to define immunological biomarkers to accurately predict vaccine efficacy have focussed mainly on systemic immune responses, and as such, studies defining correlates of protection in the genito-rectal mucosae, the primary target site for HIV entry and seeding are sparse. Clearly, difficulties in sampling and analysis of mucosal specimens, as well as their limited size have been a major deterrent in characterizing the type (mucosal antibodies, cytokines, chemokines or CTL), threshold (magnitude, depth and breadth) and viral inhibitory capacity of HIV-1 specific immune responses in the genito-rectal mucosae, where they are needed to immediately block HIV acquisition and arrest subsequent virus dissemination. Nevertheless, a few studies document the existence of HIV-specific immune responses in the genito-rectal mucosae of HIV-infected aviremic and viremic controllers, as well as in highly exposed persistently seronegative (HEPS) individuals with natural resistance to HIV-1. Some of these responses strongly correlate with protection from HIV acquisition and/or disease progression, thus providing significant clues of the ideal components of an efficacious HIV vaccine. In this article, we provide an overview of the key features of protective immune responses found in HEPS, elite and viremic controllers and discuss how these can be achieved through mucosal immunization. Inevitably, HIV vaccine development research will have to consider strategies which elicit potent antibody and cellular immune responses within the genito-rectal mucosae or induction of systemic immune cells with an inherent potential to home and persist at mucosal sites of HIV entry.

Long-term non-progressors (LTNP) were identified after 10–15 years of the epidemic, and have been the subject of intense investigation ever since. In a small minority of cases, infection with nef/3′LTR deleted attenuated viral strains allowed control over viral replication. A common feature of LTNP is the readily detected proliferation of CD4 T-cells in vitro, in response to p24. In some cases, the responding CD4 T-cells have cytotoxic effector function and may target conserved p24 epitopes, similar to the CD8 T-cells described below. LTNP may also carry much lower HIV DNA burden in key CD4 subsets, presumably resulting from lower viral replication during primary infection. Some studies, but not others, suggest that LTNP have CD4 T-cells that are relatively resistant to HIV infection in vitro. One possible mechanism may involve up-regulation of the cell cycle regulator p21/waf in CD4 T-cells from LTNP. Delayed progression in Caucasian LTNP is also partly associated with heterozygosity of the Δ32 CCR5 allele, probably through decreased expression of CCR5 co-receptor on CD4 T-cells. However, in approximately half of Caucasian LTNP, two host genotypes, namely HLA-B57 and HLA-B27, are associated with viral control. Immunodominant CD8 T-cells from these individuals target epitopes in p24 that are highly conserved, and escape mutations have significant fitness costs to the virus. Furthermore, recent studies have suggested that these CD8 T-cells from LTNP, but not from HLA-B27 or HLA-B57 progressors, can cross-react with intermediate escape mutations, preventing full escape via compensatory mutations. Humoral immunity appears to play little part in LTNP subjects, since broadly neutralizing antibodies are rare, even amongst slow progressors. Recent genome-wide comparisons between LTNP and progressors have confirmed the HLA-B57, HLA-B27, and delta32 CCR5 allelic associations, plus indicated a role for HLA-C/KIR interactions, but have not revealed any new genotypes so far. Nevertheless, it is hoped that studying the mechanisms of intracellular restriction factors, such as the recently identified SAMHD1, will lead to a better understanding of non-progression.

APOBEC3G (A3G) and APOBEC3F (A3F) reduce Vif-negative HIV-1 provirus formation and cause disabling provirus G-to-A hypermutation in vitro. However, evidence conflicts about whether they negatively impact Vif-positive HIV-1, or only enhance virus genetic diversity, in vivo. We studied peripheral blood mononuclear cells (PBMC) from 19 antiretroviral-naïve, HIV-infected adults: 12 long-term non-progressors (LTNP) and 7 non-controllers (NC). Cells from LTNP had higher A3G and A3F mRNA levels, lower provirus burden, and more A3G-hypermutated positions in provirus sequence than cells from NC. A3G mRNA level was directly associated with its Hypermutation Index (HI) and inversely associated with provirus burden. Plasma HIV-1 RNA levels were inversely associated with A3G expression levels and with HI only among subjects who had HI>1. A3G HI was not associated with provirus burden. These results indicate that A3G deaminase-dependent activity above a threshold level, and its deaminase-independent functions, contribute to decreasing Vif-positive virus replication in vivo.

Background Vpr (viral protein R) is a 96 amino acids soluble protein that is expressed late during viral replication. Recent studies have focused on the role of a mutation at position 77 that might be associated with the condition of long-term non-progression, but data are still controversial. Patients and methods Fifteen long-term non-progressors (LTNP), 19 therapy-naive HIV-1-infected patients with progressive disease (Pr), 23 HIV-1-infected patients receiving sub-optimal therapy with dual nucleoside [nucleoside reverse transcriptase inhibitor (NRTI)] therapy but efficiently controlling viral replication (STP) and 19 antiretroviral therapy multi-experienced patients with actively replicating virus (MEP) were analysed. HIV-RNA was extracted from plasma samples, the Vpr region was amplified, cloned and sequenced. The Pol gene was amplified, directly sequenced and analysed using Sequence Navigator software. Results A significantly higher prevalence of the R77Q mutation was evidenced both in LTNP (86.7%) and STP (73.9%) in comparison with Pr (42.1%) and MEP (42.1%), (P = 0.007). Comparing groups of patients with progressive disease (Pr + MEP) and groups with non-progressive disease (LTNP + STP) the probability of harbouring the R77Q mutation was significantly higher in non-progressors (odds ratio, 5.16; P = 0.001). Conclusions Our results support the hypothesis of the association of R77Q mutation in the Vpr gene with delayed progression of HIV-1 disease. R77Q does not seem to be linked to a particular viral strain but might be associated to immunologic selection. The R77Q mutation might reduce CD4+ T-cell depletion possibly affecting T-cell survival in vivo by altering the pro-apoptotic activity of Vpr.

Summary Objectives About 2–5% of HIV-1-infected subjects, defined as long-term non-progressors (LTNPs), remain immunologically stable for a long time without treatment. The factors governing this condition are known only in part, and include genetic factors. Thus, we studied 20 polymorphisms of 15 genes encoding proinflammatory and immunoregulatory cytokines, chemokines and their receptors, genes involved in apoptosis, and the gene HCP5. Methods We analyzed 47 Caucasian LTNPs infected for >9 years, compared with 131 HIV-1-infected Caucasian patients defined as ‘usual progressors'. The genotypes were determined by methods based upon PCR, and the statistical analysis was performed by univariate logistic regression. Results The well-known CCR5Δ32 del32 allele, the cell death-related TNF-α-238 A and PDCD1-7209 T alleles, and HCP5 rs2395029 G, a non-coding protein associated with the HLA-B*5701, were found positively associated with the LTNP condition. No association was observed for other single nucleotide polymorphisms (SDF-1-801, IL-10-592, MCP-1-2518, CX3CR1 V249I, CCR2V64I, RANTES-403, IL-2-330, IL-1β-511, IL-4-590, FASL IVS3nt-169, FAS-670, FAS-1377, FASL IVS2nt-124, PDCD1-7146, MMP-7-181, and MMP7-153). Conclusions The novel genetic associations between allelic variants of genes TNF-α-238 and PDCD1-7209 with the LTNP condition underline the importance of host genetic factors in the progression of HIV-1 infection and in immunological preservation.