HIV-1–Specific Immunodominant T-Cell Responses Drive the Dynamics of HIV-1 Recombination Following Superinfection

A series of HIV-1 CRF01_AE/CRF07_BC recombinants were previously found to have emerged gradually in a superinfected patient (patient LNA819). However, the extent to which T-cell responses influenced the development of these recombinants after superinfection is unclear. In this study, we undertook a recombination structure analysis of the gag, pol, and nef genes from longitudinal samples of patient LNA819. A total of 9 pol and 5 nef CRF01_AE/CRF07_BC recombinants were detected. The quasispecies makeup and the composition of the pol and nef gene recombinants changed continuously, suggestive of continuous evolution in vivo. T-cell responses targeting peptides of the primary strain and the recombination regions were screened. The results showed that Pol-LY10, Pol-RY9, and Nef-GL9 were the immunodominant epitopes. Pol-LY10 overlapped with the recombination breakpoints in multiple recombinants. For the LY10 epitope, escape from T-cell responses was mediated by both recombination with a CRF07_BC insertion carrying the T467E/T472V variants and T467N/T472V mutations originating in the CRF01_AE strain. In pol recombinants R8 and R9, the recombination breakpoints were located ~23 amino acids upstream of the RY9 epitope. The appearance of new recombination breakpoints harboring a CRF07_BC insertion carrying a R984K variant was associated with escape from RY9-specific T-cell responses. Although the Nef-GL9 epitope was located either within or 10~11 amino acids downstream of the recombination breakpoints, no variant of this epitope was observed in the nef recombinants. Instead, a F85V mutation originating in the CRF01_AE strain was the main immune escape mechanism. Understanding the cellular immune pressure on recombination is critical for monitoring the new circulating recombinant forms of HIV and designing epitope-based vaccines. Vaccines targeting antigens that are less likely to escape immune pressure by recombination and/or mutation are likely to be of benefit to patients with HIV-1.

Antigenic evolution of SARS-CoV-2 in immunocompromised hosts

The apparent lack of antigenic evolution by the Delta variant (B.1.617.2) of SARS-CoV-2 during the COVID-19 pandemic is puzzling. The combination of increasing immune pressure due to the rollout of vaccines and a relatively high number of infections following the relaxation of non-pharmaceutical interventions should have created perfect conditions for immune escape variants to evolve from the Delta lineage. Instead, the Omicron variant (B.1.1.529), which is hypothesised to have evolved in an immunocompromised individual, is the first major variant to exhibit significant immune escape following vaccination programmes and is set to become globally dominant in 2022. Here, we use a simple mathematical model to explore possible reasons why the Delta lineage did not exhibit antigenic evolution and to understand how and when immunocompromised individuals affect the emergence of immune escape variants. We show that when the pathogen does not have to cross a fitness valley for immune escape to occur, immunocompromised individuals have no qualitative effect on antigenic evolution (although they may accelerate immune escape if within-host evolutionary dynamics are faster in immunocompromised individuals). But if a fitness valley exists between immune escape variants at the between-host level, then persistent infections of immunocompromised individuals allow mutations to accumulate, therefore facilitating rather than simply speeding up antigenic evolution. Our results suggest that better global health equality, including improving access to vaccines and treatments for individuals who are immunocompromised (especially in lower- and middle-income countries), may be crucial to preventing the emergence of future immune escape variants of SARS-CoV-2.

A Pre-Vaccination Baseline of SARS-CoV-2 Genetic Surveillance and Diversity in the United States

COVID-19 vaccines were first administered on 15 December 2020, marking an important transition point for the spread of SARS-CoV-2 in the United States (U.S.). Prior to this point in time, the virus spread to an almost completely immunologically naïve population, whereas subsequently, vaccine-induced immune pressure and prior infections might be expected to influence viral evolution. Accordingly, we conducted a study to characterize the spread of SARS-CoV-2 in the U.S. pre-vaccination, investigate the depth and uniformity of genetic surveillance during this period, and measure and otherwise characterize changing viral genetic diversity, including by comparison with more recently emergent variants of concern (VOCs). In 2020, SARS-CoV-2 spread across the U.S. in three phases distinguishable by peaks in the numbers of infections and shifting geographical distributions. Virus was genetically sampled during this period at an overall rate of ~1.2%, though there was a substantial mismatch between case rates and genetic sampling nationwide. Viral genetic diversity tripled over this period but remained low in comparison to other widespread RNA virus pathogens, and although 54 amino acid changes were detected at frequencies exceeding 5%, linkage among them was not observed. Based on our collective observations, our analysis supports a targeted strategy for worldwide genetic surveillance as perhaps the most sensitive and efficient means of detecting new VOCs.

Digital gene expression analysis of NSCLC-patients reveals strong immune pressure, resulting in an immune escape under immunotherapy

Background Immune checkpoint inhibitors (ICIs) are currently one of the most promising therapy options in the field of oncology. Although the first pivotal ICI trial results were published in 2011, few biomarkers exist to predict their therapy outcome. PD-L1 expression and tumor mutational burden (TMB) were proven to be sometimes-unreliable biomarkers. We have previously suggested the analysis of processing escapes, a qualitative measurement of epitope structure alterations under immune system pressure, to provide predictive information on ICI response. Here, we sought to further validate this approach and characterize interactions with different forms of immune pressure. Methods We identified a cohort consisting of 48 patients with advanced non-small cell lung cancer (NSCLC) treated with nivolumab as ICI monotherapy. Tumor samples were subjected to targeted amplicon-based sequencing using a panel of 22 cancer-associated genes covering 98 mutational hotspots. Altered antigen processing was predicted by NetChop, and MHC binding verified by NetMHC. The NanoString nCounter® platform was utilized to provide gene expression data of 770 immune-related genes. Patient data from 408 patients with NSCLC were retrieved from The Cancer Genome Atlas (TCGA) as a validation cohort. Results The two immune escape mechanisms of PD-L1 expression (TPS score) (n = 18) and presence of altered antigen processing (n = 10) are mutually non-exclusive and can occur in the same patient (n = 6). Both mechanisms have exclusive influence on different genes and pathways, according to differential gene expression analysis and gene set enrichment analysis, respectively. Interestingly, gene expression patterns associated with altered processing were enriched in T cell and NK cell immune activity. Though both mechanisms influence different genes, they are similarly linked to increased immune activity. Conclusion Pressure from the immune system will lay the foundations for escape mechanisms, leading to acquisition of resistance under therapy. Both PD-L1 expression and altered antigen processing are induced similarly by pronounced immunoactivity but in different context. The present data help to deepen our understanding of the underlying mechanisms behind those immune escapes.

In vivo Infection Dynamics and Human Adaptive Changes of SIVsm-Derived Viral Siblings SIVmac239, SIVB670, and SIVhu in Humanized Mice as a Paralog of HIV-2 Genesis

Simian immunodeficiency virus native to sooty mangabeys (SIVsm) is believed to have given rise to HIV-2 through cross-species transmission and evolution in the human. SIVmac239 and SIVB670, pathogenic to macaques, and SIVhu, isolated from an accidental human infection, also have origins in SIVsm. With their common ancestral lineage as that of HIV-2 from the progenitor SIVsm, but with different passage history in different hosts, they provide a unique opportunity to evaluate cross-species transmission to a new host and their adaptation/evolution both in terms of potential genetic and phenotypic changes. Using humanized mice with a transplanted human system, we evaluated in vivo replication kinetics, CD4+ T cell dynamics and genetic adaptive changes during serial passage with a goal to understand their evolution under human selective immune pressure. All the three viruses readily infected hu-mice causing chronic viremia. While SIVmac and SIVB670 caused CD4+ T cell depletion during sequential passaging, SIVhu with a deletion in nef gene was found to be less pathogenic. Deep sequencing of the genomes of these viruses isolated at different times revealed numerous adaptive mutations of significance that increased in frequency during sequential passages. The ability of these viruses to infect and replicate in humanized mice provides a new small animal model to study SIVs in vivo in addition to more expensive macaques. Since SIVmac and related viruses have been indispensable in many areas of HIV pathogenesis, therapeutics and cure research, availability of this small animal hu-mouse model that is susceptible to both SIV and HIV viruses is likely to open novel avenues of investigation for comparative studies using the same host.

Polyclonal Broadly Neutralizing Antibody Activity Characterized by CD4 Binding Site and V3-Glycan Antibodies in a Subset of HIV-1 Virus Controllers

Broadly neutralizing antibodies (bNAbs), known to mediate immune control of HIV-1 infection, only develop in a small subset of HIV-1 infected individuals. Despite being traditionally associated with patients with high viral loads, bNAbs have also been observed in therapy naïve HIV-1+ patients naturally controlling virus replication [Virus Controllers (VCs)]. Thus, dissecting the bNAb response in VCs will provide key information about what constitutes an effective humoral response to natural HIV-1 infection. In this study, we identified a polyclonal bNAb response to natural HIV-1 infection targeting CD4 binding site (CD4bs), V3-glycan, gp120-gp41 interface and membrane-proximal external region (MPER) epitopes on the HIV-1 envelope (Env). The polyclonal antiviral antibody (Ab) response also included antibody-dependent cellular phagocytosis of clade AE, B and C viruses, consistent with both the Fv and Fc domain contributing to function. Sequence analysis of envs from one of the VCs revealed features consistent with potential immune pressure and virus escape from V3-glycan targeting bNAbs. Epitope mapping of the polyclonal bNAb response in VCs with bNAb activity highlighted the presence of gp120-gp41 interface and CD4bs antibody classes with similar binding profiles to known potent bNAbs. Thus, these findings reveal the induction of a broad and polyfunctional humoral response in VCs in response to natural HIV-1 infection.

Antigenic evolution of human influenza H3N2 neuraminidase is constrained by charge balancing

As one of the main influenza antigens, neuraminidase (NA) in H3N2 virus has evolved extensively for more than 50 years due to continuous immune pressure. While NA has recently emerged as an effective vaccine target, biophysical constraints on the antigenic evolution of NA remain largely elusive. Here, we apply combinatorial mutagenesis and next-generation sequencing to characterize the local fitness landscape in an antigenic region of NA in six different human H3N2 strains that were isolated around 10 years apart. The local fitness landscape correlates well among strains and the pairwise epistasis is highly conserved. Our analysis further demonstrates that local net charge governs the pairwise epistasis in this antigenic region. In addition, we show that residue coevolution in this antigenic region is correlated with the pairwise epistasis between charge states. Overall, this study demonstrates the importance of quantifying epistasis and the underlying biophysical constraint for building a model of influenza evolution.

Predicted CTL responses from pressured epitopes in SARS-CoV-2 correlate with COVID-19 severity

Heterogeneity in susceptibility among individuals to COVID-19 has been evident through the pandemic worldwide. Protective cytotoxic T lymphocyte (CTL) responses generated against pathogens in certain individuals are known to impose selection pressure on the pathogen, thus driving emergence of new variants. In this study, we focus on the role played by host genetic heterogeneity in terms of HLA-genotypes in determining differential COVID-19 severity in patients and dictating mechanisms of immune evasion adopted by SARS-CoV-2 due to the imposed immune pressure at global and cohort levels. We use bioinformatic tools for CTL epitope prediction to identify epitopes under immune pressure. Using HLA-genotype data of COVID-19 patients from a local cohort, we observe that asymptomatic individuals recognize a larger number of pressured epitopes which could facilitate emergence of mutations at these epitopic regions to overcome the protectivity they offer to the host. Based on the severity of COVID-19, we also identify HLA-alleles and epitopes that offer higher protectivity against severe disease in infected individuals. Finally, we shortlist a set of pressured and protective epitopes that represent regions in the viral proteome that are under higher immune pressure across SARS-CoV-2 variants due to the protectivity they offer. Identification of such epitopes could potentially aid in prediction of indigenous variants of SARS-CoV-2 and other pathogens, defined by the distribution of HLA-genotypes among members of a population.

ADCC-mediating non-neutralizing antibodies can exert immune pressure in early HIV-1 infection

Despite antibody-dependent cellular cytotoxicity (ADCC) responses being implicated in protection from HIV-1 infection, there is limited evidence that they control virus replication. The high mutability of HIV-1 enables the virus to rapidly adapt, and thus evidence of viral escape is a very sensitive approach to demonstrate the importance of this response. To enable us to deconvolute ADCC escape from neutralizing antibody (nAb) escape, we identified individuals soon after infection with detectable ADCC responses, but no nAb responses. We evaluated the kinetics of ADCC and nAb responses, and viral escape, in five recently HIV-1-infected individuals. In one individual we detected viruses that escaped from ADCC responses but were sensitive to nAbs. In the remaining four participants, we did not find evidence of viral evolution exclusively associated with ADCC-mediating non-neutralizing Abs (nnAbs). However, in all individuals escape from nAbs was rapid, occurred at very low titers, and in three of five cases we found evidence of viral escape before detectable nAb responses. These data show that ADCC-mediating nnAbs can drive immune escape in early infection, but that nAbs were far more effective. This suggests that if ADCC responses have a protective role, their impact is limited after systemic virus dissemination.

Spectrum of Molecular Modes of Immune Escape in Idiopathic Aplastic Anemia and Paroxysmal Nocturnal Hemoglobinuria

The pathogenesis of idiopathic aplastic anemia (IAA) involves a human leukocyte antigen (HLA)-restricted T-cell autoreactivity against unknown antigens preferentially distributed on early hematopoietic stem and progenitor cells (HSPCs). Genetically acquired GPI-anchor and HLA deficiency have been both linked to clonal immune evasion from T-cell pressure. We hypothesized that, in analogy to anti-tumor adaptive immune evasion, pathophysiology of immune escape in IAA originates together with a broader dysfunction of antigen presentation/processing machinery and immune regulatory proteins, beyond HLA molecules, as an effect of immune pressure under T-cell attack. This initial immune reaction would produce up-modulation of these pathways, ultimately promoting the acquisition of mutations and expansion of immune resistant clones. To test this hypothesis, we first performed single-cell RNAseq analysis in HSPCs in IAA patients at disease manifestation, 1 which showed signatures of dysfunction of antigen presentation machinery, with up-regulation of most of the HLA molecules, proteasome subunits and endoplasmic reticulum related organelle transporters. Strikingly, DRB1 was among the top 3 genes upregulated in IAA patients compared to controls (q-values 1.23E-35; Fig.1A), underscoring the etiological impact that antigen presentation via this locus has in the initiation of autoimmune process. Mild upregulation was also seen in DQB1 and B loci (q-values 4.7E-07 and 2.1E-10, respectively). We then studied molecular escape mechanisms by genotyping 204 IAA and PNH patients, with either a targeted or whole genome sequencing (WGS) platform. By application of a newly in-house developed bioinformatic pipeline, we detected somatic aberrations in HLA region involving both class I and II alleles in 36% of IAA patients including point mutations, frameshift insertions or deletions and copy number variations inducing allelic loss. B*14:02 and A*02:01 emerged as the most commonly mutated class I alleles with a few hotspot mutations identified, particularly in exon 1 (c.19C>T, p.R7X, Fig.1B,C), confirming previous reports. 2,3 In class II, DQB1 and DPA1 loci were frequently targeted by fine mutational events, while more complex allelic loss phenomena interested prevalently DRB1 and DQB1 loci. Those aberrations were identified at diagnosis (35%), during disease follow-up (33%) or at the time of malignant evolution (27%), with higher clonal size in specimens collected during the course of the disease (median VAF 3% [2-27%] at diagnosis, 8% [2-98%] at follow-up, and 2.2% [2.0-6.1%] at evolution). Of 41 patients with at least one HLA aberration and characterized with an extended genotypic study, only 6 harbored also >1 somatic myeloid mutation (14%), versus 30/90 (33%) not affected by somatic hits in HLA (p=.026; Fig.1D). HLA aberrant cases also showed lower number of somatic myeloid mutations (OR=0.44; p=.0262) with driver hits rarely present (Fig.1E). In terms of PIGA mutations, an increased PIGA mosaicism was observed in the HLA mutant group, underlying that both processes have similar pathophysiologic origin as a product of the immune selection pressure (OR: 1.55 [95%CI 1.1-4.2], p=.0201). We then investigated, through WGS of 53 patients, the presence of somatic mutations in other immune genes which could be triggered by immune pressure. Hence, in 47% of the cases we were able to find pathogenic or likely pathogenic hits in genes encompassing proteasome complex, vesicle trafficking, transactivators and interferon regulatory factors, including CREBBP, TAP1, CIITA, PSMC5, PSMB4 and IRF9 (Fig.1F), whose pathogenicity was computationally assessed through recently implemented somatic classifiers. 4 Those hits were not mutually exclusive neither with HLA nor with PIGA mutations, however their VAF was significantly lower compared to concurrent HLA and PIGA lesions, underscoring their lower driver potential within the immune escape environment compared to PNH and HLA-lacking clones. Altogether our results describe the diversity of molecular and immune events taking place in IAA and PNH. Our study suggests that following initial immune insult, clonal architecture of residual hematopoiesis can be dominated by multiple modes of immune escape, agonistically participating to a mechanism of "adaptive" clonal recovery, likely in opposition to the "maladaptive" malignant progression. Figure 1 Figure 1. Disclosures Maciejewski: Alexion: Consultancy; Regeneron: Consultancy; Novartis: Consultancy; Bristol Myers Squibb/Celgene: Consultancy.