Immunoglobulin G genetic variation can confound assessment of antibody levels via altered binding to detection reagents.

Objectives: Amino acid variations across more than 30 immunoglobulin (Ig) allotypes may introduce structural changes that influence recognition by anti-Ig detection reagents, consequently confounding interpretation of antibody responses, particularly in genetically diverse cohorts. Here, we assessed a panel of commercial monoclonal anti-IgG1 clones for capacity to universally recognise two dominant IgG1 haplotypes (G1m-1,3 and G1m1,17). Methods: Four commercial monoclonal anti-human IgG1 clones were assessed via ELISAs and multiplex bead-based assays for their ability to bind G1m-1,3 and G1m1,17 IgG1 variants. Detection antibodies were validated against monoclonal IgG1 allotype standards and tested for capacity to recognise antigen-specific plasma IgG1 from G1m-1,3 and G1m1,17 homozygous and heterozygous SARS-CoV-2 BNT162b2 vaccinated (n = 28) and COVID-19 convalescent (n = 44) individuals. An Fc-specific pan-IgG detection antibody corroborated differences between hinge- and Fc-specific anti-IgG1 responses. Results: Hinge-specific anti-IgG1 clone 4E3 preferentially bound G1m1,17 compared to G1m-1,3 IgG1. Consequently, SARS-CoV-2 Spike-specific IgG1 levels detected in G1m1,17/G1m1,17 BNT162b2 vaccinees appeared 9- to 17-fold higher than in G1m-1,3/G1m-1,3 vaccinees. Fc-specific IgG1 and pan-IgG detection antibodies equivalently bound G1m-1,3 and G1m1,17 IgG1 variants, and detected comparable Spike-specific IgG1 levels between haplotypes. IgG1 responses against other human coronaviruses and influenza were similarly poorly detected by 4E3 anti-IgG1 in G1m-1,3/G1m-1,3 subjects. Conclusion: Anti-IgG1 clone 4E3 confounds assessment of antibody responses in clinical cohorts owing to bias towards detection of G1m1,17 IgG1 variants. Validation of anti-Ig clones should include evaluation of binding to relevant antibody variants, particularly as the role of immunogenetics upon humoral immunity is increasingly explored in diverse populations.

Coformulation with Tattoo Ink for Immunological Assessment of Vaccine Immunogenicity in the Draining Lymph Node.

Characterization of germinal center B and T cell responses yields critical insights into vaccine immunogenicity. Nonhuman primates are a key preclinical animal model for human vaccine development, allowing both lymph node (LN) and circulating immune responses to be longitudinally sampled for correlates of vaccine efficacy. However, patterns of vaccine Ag drainage via the lymphatics after i.m. immunization can be stochastic, driving uneven deposition between lymphoid sites and between individual LN within larger clusters. To improve the accurate isolation of Ag-exposed LN during biopsies and necropsies, we developed and validated a method for coformulating candidate vaccines with tattoo ink in both mice and pigtail macaques. This method allowed for direct visual identification of vaccine-draining LN and evaluation of relevant Ag-specific B and T cell responses by flow cytometry. This approach is a significant advancement in improving the assessment of vaccine-induced immunity in highly relevant nonhuman primate models.

Templated Polymer Replica Nanoparticles to Facilitate Assessment of Material-Dependent Pharmacokinetics and Biodistribution.

Surface modification is frequently used to tailor the interactions of nanoparticles with biological systems. In many cases, the chemical nature of the treatments employed to modify the biological interface (for example attachment of hydrophilic polymers or targeting groups) is the focus of attention. However, isolation of the fundamental effects of the materials employed to modify the interface are often confounded by secondary effects imparted by the underlying substrate. Herein, we demonstrate that polymer replica particles templated from degradable mesoporous silica provide a facile means to evaluate the impact of surface modification on the biological interactions of nanomaterials, independent of the substrate. Poly(ethylene glycol) (PEG), poly(N-(2 hydroxypropyl)methacrylamide) (PHPMA), and poly(methacrylic acid) (PMA) were templated onto mesoporous silica and cross-linked and the residual particles were removed. The resulting nanoparticles, comprising interfacial polymer alone, were then investigated using a range of in vitro and in vivo tests. As expected, the PEG particles showed the best stealth properties, and these trends were consistent in both in vitro and in vivo studies. PMA particles showed the highest cell association in cell lines in vitro and were rapidly taken up by monocytes in ex vivo whole blood, properties consistent with the very high in vivo clearance subsequently seen in rats. In contrast, PHPMA particles showed rapid association with both granulocytes and monocytes in ex vivo whole blood, even though in vivo clearance was less rapid than the PMA particles. Rat studies confirmed better systemic exposure for PEG and PHPMA particles when compared to PMA particles. This study provides a new avenue for investigating material-dependent biological behaviors of polymer particles, irrespective of the properties of the underlying core, and provides insights for the selection of polymer particles for future biological applications.

The high cost of fidelity.

The notoriously low fidelity of HIV-1 replication is largely responsible for the virus's rapid mutation rate, facilitating escape from immune or drug control. The error-prone activity of the viral reverse transcriptase (RT) is predicted to be the most influential mechanism for generating mutations. The low fidelity of RT has been successfully exploited by nucleoside and nucleotide analogue reverse transcriptase inhibitors (NRTIs) that halt viral replication upon incorporation. Consequently, drug-resistant strains have arisen in which the viral RT has an increased fidelity of replication, thus reducing analogue incorporation. Higher fidelity, however, impacts on viral fitness. The appearance of compensatory mutations in combination with higher fidelity NRTI resistance mutations and the subsequent reversion of NRTI-resistant mutations upon cessation of antiretroviral treatment lend support to the notion that higher fidelity exacts a fitness cost. Potential mechanisms for reduced viral fitness are a smaller pool of mutant strains available to respond to immune or drug pressure, slower rates of replication, and a limitation to the dNTP tropism of the virus. Unraveling the relationship between replication fidelity and fitness should lead to a greater understanding of the evolution and control of HIV.

In vivo fitness costs of different Gag CD8 T-cell escape mutant simian-human immunodeficiency viruses for macaques.

The kinetics of immune escape and reversion depend upon the efficiency of CD8 cytotoxic T lymphocytes (CTL) and the fitness cost of escape mutations. Escape kinetics of three simian immunodeficiency virus Gag CTL epitopes in pigtail macaques were variable; those of KP9 and AF9 were faster than those of KW9. Kinetics of reversion of escape mutant virus to wild type upon passage to naïve major histocompatibility complex-mismatched macaques also varied. Rapid reversion occurred at KP9, gradual biphasic reversion occurred at AF9, and escape mutant KW9 virus failed to revert. The fitness impact of these mutations is KP9 > AF9 > KW9. These data provide insights into the differential utility of CTL in controlling viremia.

Rapid viral escape at an immunodominant simian-human immunodeficiency virus cytotoxic T-lymphocyte epitope exacts a dramatic fitness cost.

Escape from specific T-cell responses contributes to the progression of human immunodeficiency virus type 1 (HIV-1) infection. T-cell escape viral variants are retained following HIV-1 transmission between major histocompatibility complex (MHC)-matched individuals. However, reversion to wild type can occur following transmission to MHC-mismatched hosts in the absence of cytotoxic T-lymphocyte (CTL) pressure, due to the reduced fitness of the escape mutant virus. We estimated both the strength of immune selection and the fitness cost of escape variants by studying the rates of T-cell escape and reversion in pigtail macaques. Near-complete replacement of wild-type with T-cell escape viral variants at an immunodominant simian immunodeficiency virus Gag epitope KP9 occurred rapidly (over 7 days) following infection of pigtail macaques with SHIVSF162P3. Another challenge virus, SHIVmn229, previously serially passaged through pigtail macaques, contained a KP9 escape mutation in 40/44 clones sequenced from the challenge stock. When six KP9-responding animals were infected with this virus, the escape mutation was maintained. By contrast, in animals not responding to KP9, rapid reversion of the K165R mutation occurred over 2 weeks after infection. The rapidity of reversion to the wild-type sequence suggests a significant fitness cost of the T-cell escape mutant. Quantifying both the selection pressure exerted by CTL and the fitness costs of escape mutation has important implications for the development of CTL-based vaccine strategies.