Search NASA⌕ Search

Engineering topics

Zhang, Baoshan

Publications and source records attributed to Zhang, Baoshan.

18 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Diverse Murine Vaccinations Reveal Distinct Antibody Classes to Target Fusion Peptide and Variation in Peptide Length to Improve HIV Neutralization

The HIV-1 fusion peptide has been identified as a site for elicitation of broadly neutralizing antibodies, with prior studies demonstrating that priming with fusion peptide-based immunogens and boosting with soluble envelope (Env) trimers can elicit cross-clade HIV-1-neutralizing responses. To improve the neutralizing breadth and potency of fusion peptide-directed responses, we evaluated vaccine regimens that incorporated diverse fusion peptide-conjugates and Env trimers with variation in fusion peptide length and sequence.

Virology↗
DOE OSTI2023

Structural and immunological differences in Plasmodium falciparum sexual stage transmission-blocking vaccines comprised of Pfs25-EPA nanoparticles

Development of a malaria vaccine that blocks transmission of different parasite stages to humans and mosquitoes is considered critical for elimination efforts. A vaccine using Pfs25, a protein on the surface of zygotes and ookinetes, is under investigation as a transmission-blocking vaccine (TBV) that would interrupt parasite passage from mosquitoes to humans. The most extensively studied Pfs25 TBVs use Pichia pastoris-produced recombinant forms of Pfs25, chemically conjugated to a recombinant carrier protein, ExoProtein A (EPA). The recombinant form of Pfs25 first used in humans was identified as Pfs25H, which contained a total of 14 heterologous amino acid residues located at the amino- and carboxyl-termini including a His6 affinity tag. A second recombinant Pfs25, identified as Pfs25M, was produced to remove the heterologous amino acid residues and conjugated to EPA (Pfs25M-EPA). Here, monomeric Pfs25M was characterized biochemically and biophysically for identity, purity, and integrity including protein structure to assess its comparability with Pfs25H. Although the biological activities of Pfs25H and Pfs25M, whether generated by monomeric forms or conjugated nanoparticles, appeared similar, fine-mapping studies with two transmission-blocking monoclonal antibodies detected structural and immunological differences. In addition, evaluation of antisera generated against conjugated Pfs25H or Pfs25M nanoparticles in nonhuman primates identified polyclonal IgG that recognized these structural differences.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Structural basis of glycan276-dependent recognition by HIV-1 broadly neutralizing antibodies

Recognition of N-linked glycan at residue N276 (glycan276) at the periphery of the CD4-binding site (CD4bs) on the HIV-envelope trimer is a formidable challenge for many CD4bs-directed antibodies. To understand how this glycan can be recognized, here we isolate two lineages of glycan276-dependent CD4bs antibodies. Antibody CH540-VRC40.01 (named for donor-lineage.clone) neutralizes 81% of a panel of 208 diverse strains, while antibody CH314-VRC33.01 neutralizes 45%. Cryo-electron microscopy (cryo-EM) structures of these two antibodies and 179NC75, a previously identified glycan276-dependent CD4bs antibody, in complex with HIV-envelope trimer reveal substantially different modes of glycan276 recognition. Despite these differences, binding of glycan276-dependent antibodies maintains a glycan276 conformation similar to that observed in the absence of glycan276-binding antibodies. By contrast, glycan276-independent CD4bs antibodies, such as VRC01, displace glycan276 upon binding. These results provide a foundation for understanding antibody recognition of glycan276 and suggest its presence may be crucial for priming immunogens seeking to initiate broad CD4bs recognition.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structural basis of LAIR1 targeting by polymorphic Plasmodium RIFINs

RIFIN, a large family of Plasmodium variant surface antigens, plays a crucial role in malaria pathogenesis by mediating immune suppression through activation of inhibitory receptors such as LAIR1, and antibodies with LAIR1 inserts have been identified that bind infected erythrocytes through RIFIN. However, details of RIFIN-mediated LAIR1 recognition and receptor activation have been unclear. Here, we use negative-stain EM to define the architecture of LAIR1-inserted antibodies and determine crystal structures of RIFIN-variable 2 (V2) domain in complex with a LAIR1 domain. These structures reveal the LAIR1-binding region of RIFIN to be hydrophobic and membrane-distal, to exhibit extensive structural diversity, and to interact with RIFIN-V2 in a one-to-one fashion. Through structural and sequence analysis of various LAIR1 constructs, we identify essential elements of RIFIN-binding on LAIR1. Furthermore, a structure-derived LAIR1-binding sequence signature ascertained >20 LAIR1-binding RIFINs, including some from P. falciparum field strains and Plasmodium species infecting gorillas and chimpanzees.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structures of HIV-1 Neutralizing Antibody 10E8 Delineate the Mechanistic Basis of Its Multi-Peak Behavior on Size-Exclusion Chromatography

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

Structure and function of a malaria transmission blocking vaccine targeting Pfs230 and Pfs230-Pfs48/45 proteins

Proteins Pfs230 and Pfs48/45 are Plasmodium falciparum transmission-blocking (TB) vaccine candidates that form a membrane-bound protein complex on gametes. The biological role of Pfs230 or the Pfs230-Pfs48/45 complex remains poorly understood. Here, we present the crystal structure of recombinant Pfs230 domain 1 (Pfs230D1M), a 6-cysteine domain, in complex with the Fab fragment of a TB monoclonal antibody (mAb) 4F12. We observed the arrangement of Pfs230 on the surface of macrogametes differed from that on microgametes, and that Pfs230, with no known membrane anchor, may exist on the membrane surface in the absence of Pfs48/45. 4F12 appears to sterically interfere with Pfs230 function. Combining mAbs against different epitopes of Pfs230D1 or of Pfs230D1 and Pfs48/45, significantly increased TB activity. These studies elucidate a mechanism of action of the Pfs230D1 vaccine, model the functional activity induced by a polyclonal antibody response and support the development of TB vaccines targeting Pfs230D1 and Pfs230D1-Pfs48/45.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2020

VRC34-Antibody Lineage Development Reveals How a Required Rare Mutation Shapes the Maturation of a Broad HIV-Neutralizing Lineage

Rare mutations have been proposed to restrict the development of broadly neutralizing antibodies against HIV-1, but this has not been explicitly demonstrated. We hypothesized that such rare mutations might be identified by comparing broadly neutralizing and non-broadly neutralizing branches of an antibody-developmental tree. Because sequences of antibodies isolated from the fusion peptide (FP)-targeting VRC34-antibody lineage suggested it might be suitable for such rare mutation analysis, we carried out next-generation sequencing (NGS) on B cell transcripts from donor N123, the source of the VRC34 lineage, and functionally and structurally characterized inferred intermediates along broadly neutralizing and poorly neutralizing developmental branches. The broadly neutralizing VRC34.01 branch required the rare heavy-chain mutation Y33P to bind FP, whereas the early bifurcated VRC34.05 branch did not require this rare mutation and evolved less breadth. Our results demonstrate how a required rare mutation can restrict development and shape the maturation of a broad HIV-1-neutralizing antibody lineage.

59 BASIC BIOLOGICAL SCIENCES↗