Characteristics of a PHD Finger Subtype.

Although the plant homeodomain (PHD) finger superfamily is known for its site-specific readouts of histone tails, the origins of the mechanistic differences in histone H3 readout by different PHD subtypes remain less clear. We show that sequences containing the xCDxCDx motif in the PHD treble clef (xCDxCDx-PHD) constitute a distinct subtype, based on the following observations: (i) the amino acid composition of the binding site is strikingly different from other subtypes due to position-specific enrichment of negatively charged and bulky nonpolar residues, (ii) the binding site positions are mutually and positively correlated, and this correlation is absent in other subtypes, and (iii) there are only small structural deviations, despite low sequence similarity. The xCDxCDx-PHD constitutes ∼20% of the PHD family, and the double PHD fingers (DPFs) are 10% of the total number of xCDxCDx-PHDs. This subtype originated early in the evolution of eukaryotes but has diversified within the metazoan lineage. Despite sequence diversification, the positions of the enriched nonpolar residues, in particular, show very small structural deviations, suggesting critical contributions of nonpolar residues in the binding mechanism of this subtype. Using mutagenesis, we probed the contributions of the binding-site positions enriched in nonpolar residues in four xCDxCDx-PHD proteins and found that they contribute to the tight packing of the H3 residues. This effect may potentially be exploited, as we observed affinity enhancement upon substituting a bulky nonpolar residue at the same binding site in another histone reader. Overall, we present a detailed characterization of PHD subtypes.

Assessing the functional roles of coevolving PHD finger residues.

Although in silico folding based on coevolving residue constraints in the deep-learning era has transformed protein structure prediction, the contributions of coevolving residues to protein folding, stability, and other functions in physical contexts remain to be clarified and experimentally validated. Herein, the PHD finger module, a well-known histone reader with distinct subtypes containing subtype-specific coevolving residues, was used as a model to experimentally assess the contributions of coevolving residues and to clarify their specific roles. The results of the assessment, including proteolysis and thermal unfolding of wildtype and mutant proteins, suggested that coevolving residues have varying contributions, despite their large in silico constraints. Residue positions with large constraints were found to contribute to stability in one subtype but not others. Computational sequence design and generative model-based energy estimates of individual structures were also implemented to complement the experimental assessment. Sequence design and energy estimates distinguish coevolving residues that contribute to folding from those that do not. The results of proteolytic analysis of mutations at positions contributing to folding were consistent with those suggested by sequence design and energy estimation. Thus, we report a comprehensive assessment of the contributions of coevolving residues, as well as a strategy based on a combination of approaches that should enable detailed understanding of the residue contributions in other large protein families.

Determination of Binding Affinity of Antibodies to HIV-1 Recombinant Envelope Glycoproteins, Pseudoviruses, Infectious Molecular Clones, and Cell-Expressed Trimeric gp160 Using Microscale Thermophoresis.

Developing a preventative vaccine for HIV-1 has been a global priority. The elicitation of broadly neutralizing antibodies (bNAbs) against a broad range of HIV-1 envelopes (Envs) from various strains appears to be a critical requirement for an efficacious HIV-1 vaccine. To understand their ability to neutralize HIV-1, it is important to characterize the binding characteristics of bNAbs. Our work is the first to utilize microscale thermophoresis (MST), a rapid, economical, and flexible in-solution temperature gradient method to quantitatively determine the binding affinities of bNAbs and non-neutralizing monoclonal antibodies (mAbs) to HIV-1 recombinant envelope monomer and trimer proteins of different subtypes, pseudoviruses (PVs), infectious molecular clones (IMCs), and cells expressing the trimer. Our results demonstrate that the binding affinities were subtype-dependent. The bNAbs exhibited a higher affinity to IMCs compared to PVs and recombinant proteins. The bNAbs and mAbs bound with high affinity to native-like gp160 trimers expressed on the surface of CEM cells compared to soluble recombinant proteins. Interesting differences were seen with V2-specific mAbs. Although they recognize linear epitopes, one of the antibodies also bound to the Envs on PVs, IMCs, and a recombinant trimer protein, suggesting that the epitope was not occluded. The identification of epitopes on the envelope surface that can bind to high affinity mAbs could be useful for designing HIV-1 vaccines and for down-selecting vaccine candidates that can induce high affinity antibodies to the HIV-1 envelope in their native conformation.

Cholera toxin B scaffolded, focused SIV V2 epitope elicits antibodies that influence the risk of SIVmac251 acquisition in macaques.

Introduction: An efficacious HIV vaccine will need to elicit a complex package of innate, humoral, and cellular immune responses. This complex package of responses to vaccine candidates has been studied and yielded important results, yet it has been a recurring challenge to determine the magnitude and protective effect of specific in vivo immune responses in isolation. We therefore designed a single, viral-spike-apical, epitope-focused V2 loop immunogen to reveal individual vaccine-elicited immune factors that contribute to protection against HIV/SIV. Method: We generated a novel vaccine by incorporating the V2 loop B-cell epitope in the cholera toxin B (CTB) scaffold and compared two new immunization regimens to a historically protective 'standard' vaccine regimen (SVR) consisting of 2xDNA prime boosted with 2xALVAC-SIV and 1xΔV1gp120. We immunized a cohort of macaques with 5xCTB-V2c vaccine+alum intramuscularly simultaneously with topical intrarectal vaccination of CTB-V2c vaccine without alum (5xCTB-V2/alum). In a second group, we tested a modified version of the SVR consisting of 2xDNA prime and boosted with 1xALVAC-SIV and 2xALVAC-SIV+CTB-V2/alum, (DA/CTB-V2c/alum). Results: In the absence of any other anti-viral antibodies, V2c epitope was highly immunogenic when incorporated in the CTB scaffold and generated highly functional anti-V2c antibodies in the vaccinated animals. 5xCTB-V2c/alum vaccination mediated non-neutralizing ADCC activity and efferocytosis, but produced low avidity, trogocytosis, and no neutralization of tier 1 virus. Furthermore, DA/CTB-V2c/alum vaccination also generated lower total ADCC activity, avidity, and neutralization compared to the SVR. These data suggest that the ΔV1gp120 boost in the SVR yielded more favorable immune responses than its CTB-V2c counterpart. Vaccination with the SVR generates CCR5- α4ß7+CD4+ Th1, Th2, and Th17 cells, which are less likely to be infected by SIV/HIV and likely contributed to the protection afforded in this regimen. The 5xCTB-V2c/alum regimen likewise elicited higher circulating CCR5- α4ß7+ CD4+ T cells and mucosal α4ß7+ CD4+ T cells compared to the DA/CTB-V2c/alum regimen, whereas the first cell type was associated with reduced risk of viral acquisition. Conclusion: Taken together, these data suggest that individual viral spike B-cell epitopes can be highly immunogenic and functional as isolated immunogens, although they might not be sufficient on their own to provide full protection against HIV/SIV infection.

SARS-CoV-2 spike-ferritin-nanoparticle adjuvanted with ALFQ induces long-lived plasma cells and cross-neutralizing antibodies.

This study demonstrates the impact of adjuvant on the development of T follicular helper (Tfh) and B cells, and their influence on antibody responses in mice vaccinated with SARS-CoV-2-spike-ferritin-nanoparticle (SpFN) adjuvanted with either Army Liposome Formulation containing QS-21 (SpFN + ALFQ) or Alhydrogel® (SpFN + AH). SpFN + ALFQ increased the size and frequency of germinal center (GC) B cells in the vaccine-draining lymph nodes and increased the frequency of antigen-specific naive B cells. A single vaccination with SpFN + ALFQ resulted in a higher frequency of IL-21-producing-spike-specific Tfh and GC B cells in the draining lymph nodes and spleen, S-2P protein-specific IgM and IgG antibodies, and elicitation of robust cross-neutralizing antibodies against SARS-CoV-2 variants as early as day 7, which was enhanced by a second vaccination. This was associated with the generation of high titer, high avidity binding antibodies. The third vaccination with SpFN + ALFQ elicited high levels of neutralizing antibodies against the Omicron variant. No cross-neutralizing antibodies against Omicron were induced with SpFN + AH. These findings highlight the importance of ALFQ in orchestrating early induction of antigen-specific Tfh and GC B cell responses and long-lived plasma cells in the bone marrow. The early engagement of S-2P specific naive B cells and high titer IgM antibodies shape the development of long-term neutralization breadth.