Elevated genetic risk for multiple sclerosis emerged in steppe pastoralist populations.

Multiple sclerosis (MS) is a neuro-inflammatory and neurodegenerative disease that is most prevalent in Northern Europe. Although it is known that inherited risk for MS is located within or in close proximity to immune-related genes, it is unknown when, where and how this genetic risk originated1. Here, by using a large ancient genome dataset from the Mesolithic period to the Bronze Age2, along with new Medieval and post-Medieval genomes, we show that the genetic risk for MS rose among pastoralists from the Pontic steppe and was brought into Europe by the Yamnaya-related migration approximately 5,000 years ago. We further show that these MS-associated immunogenetic variants underwent positive selection both within the steppe population and later in Europe, probably driven by pathogenic challenges coinciding with changes in diet, lifestyle and population density. This study highlights the critical importance of the Neolithic period and Bronze Age as determinants of modern immune responses and their subsequent effect on the risk of developing MS in a changing environment.

The selection landscape and genetic legacy of ancient Eurasians.

The Holocene (beginning around 12,000 years ago) encompassed some of the most significant changes in human evolution, with far-reaching consequences for the dietary, physical and mental health of present-day populations. Using a dataset of more than 1,600 imputed ancient genomes1, we modelled the selection landscape during the transition from hunting and gathering, to farming and pastoralism across West Eurasia. We identify key selection signals related to metabolism, including that selection at the FADS cluster began earlier than previously reported and that selection near the LCT locus predates the emergence of the lactase persistence allele by thousands of years. We also find strong selection in the HLA region, possibly due to increased exposure to pathogens during the Bronze Age. Using ancient individuals to infer local ancestry tracts in over 400,000 samples from the UK Biobank, we identify widespread differences in the distribution of Mesolithic, Neolithic and Bronze Age ancestries across Eurasia. By calculating ancestry-specific polygenic risk scores, we show that height differences between Northern and Southern Europe are associated with differential Steppe ancestry, rather than selection, and that risk alleles for mood-related phenotypes are enriched for Neolithic farmer ancestry, whereas risk alleles for diabetes and Alzheimer's disease are enriched for Western hunter-gatherer ancestry. Our results indicate that ancient selection and migration were large contributors to the distribution of phenotypic diversity in present-day Europeans.

Antigen processing influences HIV-specific cytotoxic T lymphocyte immunodominance.

Although cytotoxic T lymphocytes (CTLs) in people infected with human immunodeficiency virus type 1 can potentially target multiple virus epitopes, the same few are recognized repeatedly. We show here that CTL immunodominance in regions of the human immunodeficiency virus type 1 group-associated antigen proteins p17 and p24 correlated with epitope abundance, which was strongly influenced by proteasomal digestion profiles, affinity for the transporter protein TAP, and trimming mediated by the endoplasmatic reticulum aminopeptidase ERAAP, and was moderately influenced by HLA affinity. Structural and functional analyses demonstrated that proteasomal cleavage 'preferences' modulated the number and length of epitope-containing peptides, thereby affecting the response avidity and clonality of T cells. Cleavage patterns were affected by both flanking and intraepitope CTL-escape mutations. Our analyses show that antigen processing shapes CTL response hierarchies and that viral evolution modifies cleavage patterns and suggest strategies for in vitro vaccine optimization.

Possible footprints of APOBEC3F and/or other APOBEC3 deaminases, but not APOBEC3G, on HIV-1 from patients with acute/early and chronic infections.

UNLABELLED: Members of the apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like-3 (APOBEC3) innate cellular cytidine deaminase family, particularly APOBEC3F and APOBEC3G, can cause extensive and lethal G-to-A mutations in HIV-1 plus-strand DNA (termed hypermutation). It is unclear if APOBEC3-induced mutations in vivo are always lethal or can occur at sublethal levels that increase HIV-1 diversification and viral adaptation to the host. The viral accessory protein Vif counteracts APOBEC3 activity by binding to APOBEC3 and promoting proteasome degradation; however, the efficiency of this interaction varies, since a range of hypermutation frequencies are observed in HIV-1 patient DNA. Therefore, we examined "footprints" of APOBEC3G and APOBEC3F activity in longitudinal HIV-1 RNA pol sequences from approximately 3,000 chronically infected patients by determining whether G-to-A mutations occurred in motifs that were favored or disfavored by these deaminases. G-to-A mutations were more frequent in APOBEC3G-disfavored than in APOBEC3G-favored contexts. In contrast, mutations in APOBEC3F-disfavored contexts were relatively rare, whereas mutations in contexts favoring APOBEC3F (and possibly other deaminases) occurred 16% more often than average G-to-A mutations. These results were supported by analyses of >500 HIV-1 env sequences from acute/early infection. IMPORTANCE: Collectively, our results suggest that APOBEC3G-induced mutagenesis is lethal to HIV-1, whereas mutagenesis caused by APOBEC3F and/or other deaminases may result in sublethal mutations that might facilitate viral diversification. Therefore, Vif-specific cytotoxic T lymphocyte (CTL) responses and drugs that manipulate the interplay between Vif and APOBEC3 may have beneficial or detrimental clinical effects depending on how they affect the binding of Vif to various members of the APOBEC3 family.

APOBEC3G-induced hypermutation of human immunodeficiency virus type-1 is typically a discrete "all or nothing" phenomenon.

The rapid evolution of Human Immunodeficiency Virus (HIV-1) allows studies of ongoing host-pathogen interactions. One key selective host factor is APOBEC3G (hA3G) that can cause extensive and inactivating Guanosine-to-Adenosine (G-to-A) mutation on HIV plus-strand DNA (termed hypermutation). HIV can inhibit this innate anti-viral defense through binding of the viral protein Vif to hA3G, but binding efficiency varies and hypermutation frequencies fluctuate in patients. A pivotal question is whether hA3G-induced G-to-A mutation is always lethal to the virus or if it may occur at sub-lethal frequencies that could increase viral diversification. We show in vitro that limiting-levels of hA3G-activity (i.e. when only a single hA3G-unit is likely to act on HIV) produce hypermutation frequencies similar to those in patients and demonstrate in silico that potentially non-lethal G-to-A mutation rates are ∼10-fold lower than the lowest observed hypermutation levels in vitro and in vivo. Our results suggest that even a single incorporated hA3G-unit is likely to cause extensive and inactivating levels of HIV hypermutation and that hypermutation therefore is typically a discrete "all or nothing" phenomenon. Thus, therapeutic measures that inhibit the interaction between Vif and hA3G will likely not increase virus diversification but expand the fraction of hypermutated proviruses within the infected host.

Rapid antigen processing and presentation of a protective and immunodominant HLA-B*27-restricted hepatitis C virus-specific CD8+ T-cell epitope.

HLA-B*27 exerts protective effects in hepatitis C virus (HCV) and human immunodeficiency virus (HIV) infections. While the immunological and virological features of HLA-B*27-mediated protection are not fully understood, there is growing evidence that the presentation of specific immunodominant HLA-B*27-restricted CD8+ T-cell epitopes contributes to this phenomenon in both infections. Indeed, protection can be linked to single immunodominant CD8+ T-cell epitopes and functional constraints on escape mutations within these epitopes. To better define the immunological mechanisms underlying HLA-B*27-mediated protection in HCV infection, we analyzed the functional avidity, functional profile, antiviral efficacy and naïve precursor frequency of CD8+ T cells targeting the immunodominant HLA-B*27-restricted HCV-specific epitope as well as its antigen processing and presentation. For comparison, HLA-A*02-restricted HCV-specific epitopes were analyzed. The HLA-B*27-restricted CD8+ T-cell epitope was not superior to epitopes restricted by HLA-A*02 when considering the functional avidity, functional profile, antiviral efficacy or naïve precursor frequency. However, the peptide region containing the HLA-B*27-restricted epitope was degraded extremely fast by both the constitutive proteasome and the immunoproteasome. This efficient proteasomal processing that could be blocked by proteasome inhibitors was highly dependent on the hydrophobic regions flanking the epitope and led to rapid and abundant presentation of the epitope on the cell surface of antigen presenting cells. Our data suggest that rapid antigen processing may be a key immunological feature of this protective and immunodominant HLA-B*27-restricted HCV-specific epitope.

Acute Inflammatory Diseases of the Central Nervous System After SARS-CoV-2 Vaccination.

BACKGROUND AND OBJECTIVES: Acute inflammatory CNS diseases include neuromyelitis optica spectrum disorders (NMOSDs) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD). Both MOGAD and acute disseminated encephalomyelitis (ADEM) have been reported after vaccination. Consequently, the mass SARS-CoV-2 vaccination program could result in increased rates of these conditions. We described the features of patients presenting with new acute CNS demyelination resembling NMOSDs or MOGAD within 8 weeks of SARS-CoV-2 vaccination. METHODS: The study included a prospective case series of patients referred to highly specialized NMOSD services in the UK from the introduction of SARS-CoV-2 vaccination program up to May 2022. Twenty-five patients presented with new optic neuritis (ON) and/or transverse myelitis (TM) ± other CNS inflammation within 8 weeks of vaccination with either AstraZeneca (ChAdOx1S) or Pfizer (BNT162b2) vaccines. Their clinical records and paraclinical investigations including MRI scans were reviewed. Serologic testing for antibodies to myelin oligodendrocyte glycoprotein (MOG) and aquaporin 4 (AQP4) was performed using live cell-based assays. Patients' outcomes were graded good, moderate, or poor based on the last clinical assessment. RESULTS: Of 25 patients identified (median age 38 years, 14 female), 12 (48%) had MOG antibodies (MOGIgG+), 2 (8%) had aquaporin 4 antibodies (AQP4IgG+), and 11 (44%) had neither. Twelve of 14 (86%) antibody-positive patients received the ChAdOx1S vaccine. MOGIgG+ patients presented most commonly with TM (10/12, 83%), frequently in combination with ADEM-like brain/brainstem lesions (6/12, 50%). Transverse myelitis was longitudinally extensive in 7 of the 10 patients. A peak in new MOGAD cases in Spring 2021 was attributable to postvaccine cases. Both AQP4IgG+ patients presented with brain lesions and TM. Four of 6 (67%) seronegative ChAdOx1S recipients experienced longitudinally extensive TM (LETM) compared with 1 of 5 (20%) of the BNT162b2 group, and facial nerve inflammation was reported only in ChAdOx1S recipients (2/5, 40%). Guillain-Barre syndrome was confirmed in 1 seronegative ChAdOx1S recipient and suspected in another. DISCUSSION: ChAdOx1S was associated with 12/14 antibody-positive cases, the majority MOGAD. MOGAD patients presented atypically, only 2 with isolated ON (1 after BNT162b2 vaccine) but with frequent ADEM-like brain lesions and LETM. Within the seronegative group, phenotypic differences were observed between ChAdOx1S and BNT162b2 recipients. These observations might support a causative role of the ChAdOx1S vaccine in inflammatory CNS disease and particularly MOGAD. Further study of this cohort could provide insights into vaccine-associated immunopathology.

AIDS restriction HLA allotypes target distinct intervals of HIV-1 pathogenesis.

An effective acquired immune response to infectious agents mediated by HLA-restricted T-cell recognition can target different stages of disease pathogenesis. We show here that three distinct HLA alleles known to alter the overall rate of AIDS progression act during distinct intervals after HIV-1 infection. The discrete timing of HLA allele influence suggests alternative functional mechanisms in immune defense against this dynamic and chronic immunosuppressive disease.

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.

A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR.

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

HIV-1 p24Gag adaptation to modern and archaic HLA-allele frequency differences in ethnic groups contributes to viral subtype diversification.

Pathogen-driven selection and past interbreeding with archaic human lineages have resulted in differences in human leukocyte antigen (HLA)-allele frequencies between modern human populations. Whether or not this variation affects pathogen subtype diversification is unknown. Here we show a strong positive correlation between ethnic diversity in African countries and both human immunodeficiency virus (HIV)-1 p24gag and subtype diversity. We demonstrate that ethnic HLA-allele differences between populations have influenced HIV-1 subtype diversification as the virus adapted to escape common antiviral immune responses. The evolution of HIV Subtype B (HIV-B), which does not appear to be indigenous to Africa, is strongly affected by immune responses associated with Eurasian HLA variants acquired through adaptive introgression from Neanderthals and Denisovans. Furthermore, we show that the increasing and disproportionate number of HIV-infections among African Americans in the USA drive HIV-B evolution towards an Africa-centric HIV-1 state. Similar adaptation of other pathogens to HLA variants common in affected populations is likely.

Conserved footprints of APOBEC3G on Hypermutated human immunodeficiency virus type 1 and human endogenous retrovirus HERV-K(HML2) sequences.

The human polynucleotide cytidine deaminases APOBEC3G (hA3G) and APOBEC3F (hA3F) are antiviral restriction factors capable of inducing extensive plus-strand guanine-to-adenine (G-to-A) hypermutation in a variety of retroviruses and retroelements, including human immunodeficiency virus type 1 (HIV-1). They differ in target specificity, favoring plus-strand 5'GG and 5'GA dinucleotide motifs, respectively. To characterize their mutational preferences in detail, we analyzed single-copy, near-full-length HIV-1 proviruses which had been hypermutated in vitro by hA3G or hA3F. hA3-induced G-to-A mutation rates were significantly influenced by the wider sequence context of the target G. Moreover, hA3G, and to a lesser extent hA3F, displayed clear tetranucleotide preference hierarchies, irrespective of the genomic region examined and overall hypermutation rate. We similarly analyzed patient-derived hypermutated HIV-1 genomes using a new method for estimating reference sequences. The majority of these, regardless of subtype, carried signatures of hypermutation that strongly correlated with those induced in vitro by hA3G. Analysis of genome-wide hA3-induced mutational profiles confirmed that hypermutation levels were reduced downstream of the polypurine tracts. Additionally, while hA3G mutations were found throughout the genome, hA3F often intensely mutated shorter regions, the locations of which varied between proviruses. We extended our analysis to human endogenous retroviruses (HERVs) from the HERV-K(HML2) family, finding two elements that carried clear footprints of hA3G activity. This constitutes the most direct evidence to date for hA3G activity in the context of natural HERV infections, demonstrating the involvement of this restriction factor in defense against retroviral attacks over millions of years of human evolution.

HIV-infected sex workers with beneficial HLA-variants are potential hubs for selection of HIV-1 recombinants that may affect disease progression.

Cytotoxic T lymphocyte (CTL) responses against the HIV Gag protein are associated with lowering viremia; however, immune control is undermined by viral escape mutations. The rapid viral mutation rate is a key factor, but recombination may also contribute. We hypothesized that CTL responses drive the outgrowth of unique intra-patient HIV-recombinants (URFs) and examined gag sequences from a Kenyan sex worker cohort. We determined whether patients with HLA variants associated with effective CTL responses (beneficial HLA variants) were more likely to carry URFs and, if so, examined whether they progressed more rapidly than patients with beneficial HLA-variants who did not carry URFs. Women with beneficial HLA-variants (12/52) were more likely to carry URFs than those without beneficial HLA variants (3/61) (p < 0.0055; odds ratio = 5.7). Beneficial HLA variants were primarily found in slow/standard progressors in the URF group, whereas they predominated in long-term non-progressors/survivors in the remaining cohort (p = 0.0377). The URFs may sometimes spread and become circulating recombinant forms (CRFs) of HIV and local CRF fragments were over-represented in the URF sequences (p < 0.0001). Collectively, our results suggest that CTL-responses associated with beneficial HLA variants likely drive the outgrowth of URFs that might reduce the positive effect of these CTL responses on disease progression.

Longitudinal and cross-sectional studies of HIV-1 RNA and DNA loads in blood and the female genital tract.

OBJECTIVE: To examine if correlates of HIV-1 genital shedding in cross-sectional studies can be used to determine the risk of shedding in individual HIV-1-positive women. STUDY DESIGN: Longitudinal samples from blood and cervix were obtained from 18 HIV-1 infected women, and HIV-1 RNA and cell-associated DNA virus, and beta-chemokine levels, were measured. Associations between variables were analyzed at both individual and group level. RESULTS: The variation over time was 2.9-, 2.1-, and 2.3-fold in plasma RNA, PBMC DNA and cervical RNA load, respectively, and reached 6.2-fold in cervical DNA load. Differences were observed between associations in individual- and group-level comparisons, suggesting that a separate reservoir of HIV replication may exist in the genital tract of some women, which is influenced by local environmental factors. CONCLUSIONS: Our study underscores the importance of caution during contact with genital fluids at all stages of infection and disease regardless of treatment and HIV-1 blood loads.

HIV-1 adaptation to antigen processing results in population-level immune evasion and affects subtype diversification.

The recent HIV-1 vaccine failures highlight the need to better understand virus-host interactions. One key question is why CD8(+) T cell responses to two HIV-Gag regions are uniquely associated with delayed disease progression only in patients expressing a few rare HLA class I variants when these regions encode epitopes presented by ~30 more common HLA variants. By combining epitope processing and computational analyses of the two HIV subtypes responsible for ~60% of worldwide infections, we identified a hitherto unrecognized adaptation to the antigen-processing machinery through substitutions at subtype-specific motifs. Multiple HLA variants presenting epitopes situated next to a given subtype-specific motif drive selection at this subtype-specific position, and epitope abundances correlate inversely with the HLA frequency distribution in affected populations. This adaptation reflects the sum of intrapatient adaptations, is predictable, facilitates viral subtype diversification, and increases global HIV diversity. Because low epitope abundance is associated with infrequent and weak T cell responses, this most likely results in both population-level immune evasion and inadequate responses in most people vaccinated with natural HIV-1 sequence constructs. Our results suggest that artificial sequence modifications at subtype-specific positions in vitro could refocus and reverse the poor immunogenicity of HIV proteins.

Sequential broadening of CTL responses in early HIV-1 infection is associated with viral escape.

BACKGROUND: Antigen-specific CTL responses are thought to play a central role in containment of HIV-1 infection, but no consistent correlation has been found between the magnitude and/or breadth of response and viral load changes during disease progression. METHODS AND FINDINGS: We undertook a detailed investigation of longitudinal CTL responses and HIV-1 evolution beginning with primary infection in 11 untreated HLA-A2 positive individuals. A subset of patients developed broad responses, which selected for consensus B epitope variants in Gag, Pol, and Nef, suggesting CTL-induced adaptation of HIV-1 at the population level. The patients who developed viral escape mutations and broad autologous CTL responses over time had a significantly higher increase in viral load during the first year of infection compared to those who did not develop viral escape mutations. CONCLUSIONS: A continuous dynamic development of CTL responses was associated with viral escape from temporarily effective immune responses. Our results suggest that broad CTL responses often represent footprints left by viral CTL escape rather than effective immune control, and help explain earlier findings that fail to show an association between breadth of CTL responses and viral load. Our results also demonstrate that CTL pressures help to maintain certain elements of consensus viral sequence, which likely represent viral escape from common HLA-restricted CTL responses. The ability of HIV to evolve to escape CTL responses restricted by a common HLA type highlights the challenges posed to development of an effective CTL-based vaccine.

Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope.

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Limited protective effect of the CCR5Delta32/CCR5Delta32 genotype on human immunodeficiency virus infection incidence in a cohort of patients with hemophilia and selection for genotypic X4 virus.

The relationship among CCR5 genotype, cytomegalovirus infection, and disease progression and death was studied among 159 human immunodeficiency virus (HIV)-infected patients with hemophilia. One patient (0.6%) had the CCR5Delta32/CCR5Delta32 genotype (which occurs in approximately 2% of the Scandinavian population) and a rapid disease course. His HIV V3 region contained genotypic features attributable to X4 virus and resembled functionally verified X4 virus and virus from patients treated with a CD4 cell-stimulating drug, tucaresol. Age-related differences in disease progression rate and survival time were seen for CCR5/CCR5 patients. Surprisingly, no protective effect of the CCR5/CCR5Delta32 genotype on disease progression or survival was seen for children but was evident for adults. Age group-related immunologic differences might explain this variation, and transmission route and/or viral phenotype variation within donor virus may be related to the limited protection of the CCR5Delta32/CCR5Delta32 genotype. Sequence comparisons indicate that X4 virus can be selected in vivo due to either absence of CCR5 receptors or relative increase of CXCR4 receptors.