1008. Presence of Antibody Dependent Cell Cytotoxicity (ADCC) Functional Antibodies that Target a Complex Gp41 Epitope Correlates with Long-term Non-progression and ADCC is Maintained with Mutants Using Germline Heavy Chain Variable Gene Sequence of VH1-02 Gene

Abstract Background Recent data supports that improved qualitative antibody responses correlate with elite controllers (EC) of HIV. As ADCC has been associated with protection in vaccine studies, thorough exploration of antibodies that facilitate ADCC is warranted. In studies on monoclonal antibodies from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated antibodies against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing antibodies against HIV, members of these antibodies, termed group 76C antibodies, did not exhibit broad neutralization. Methods Our goal was to characterize the non-neutralizing functions of antibodies of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these antibodies by comparison to their predicted common ancestor. Serum samples were obtained from HIV+ clinical groups: EC, LTNP, stable CD4 counts on therapy, and those off therapy. Results In antibody/serum competition assays, comparison to VRC01 which also uses VH1-02, showed that antibodies targeting the 76C group epitope were enriched in LTNPs. We then show recombinant antibodies of 76C members 6F5 and 6F11 both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc also shows comparable ADCC to 6F5 and 6F11 on both clade B and C constructs. Common ancestor antibodies maintain function and these types of antibodies correlate to a non-progressive clinical state. (A) Serum from long-term non-progressors (LTNPs) compared to serum from a group of HIV infected with lower CD4 levels as a control for viral load were used to compete against biotinylated CD4 binding site (VRC01) and 76C Gp41 conformational epitope (6F11) targeting antibodies. Serum dilutions were chosen to align means near 50%. Means with 95% confidence intervals are shown. (B) Monoclonal antibody 76Canc was created using the germline sequence of the heavy chain variable region with the CDR3 and light chain of 76C member. Antibody dependent cell cytotoxicity flow cytometric based assays were performed using gp41 proteins from clade B (MN) and clade C (ZA1197). Conclusion Certain antibodies present early on in infection may contribute to overall clinical course. Variable gene germline sequences that support functional activity against HIV could be targeted in vaccine regimens. Disclosures All Authors: No reported disclosures

. Univariable and multivariable analysis of factors related to pneumonia Conclusion. The association between increased ompA expression in A. baumannii and the development of pneumonia was not statistically significant after adjusting for patient factors. However, the relatively high expression of ompA in pneumonia patients and their association with increased mortality suggests the need for larger-scale prospective studies to draw a conclusion.
Disclosures. All Authors: No reported disclosures

Session: P-56. Microbial Pathogenesis
Background. Hypermutator (HM) bacteria exhibit high spontaneous mutation rates due to DNA mismatch repair (MMR) gene mutations, which may facilitate antibiotic resistance. HM is best described for chronic infections or colonization, in particular with P. aeruginosa. HM K. pneumoniae (KP) and carbapenem resistant Enterobacterales (CRE) are rarely studied.
Methods. Longitudinal isolates from 5 patients (pts) with long-term ST258 CRKP infections (median: 1.4 yr, 0.5-4.1 yr) underwent Illumina HiSeq whole genome sequencing. Strains from 1 pt were tested for HM and resistance. Mutant strains were created by complementation and CRISPR.
SNP matrix of 11 clinical isolates from a single patient with recurrent KPC-Kp infections The first 6 isolates were recovered within 6 months of transplant (Tinitial). The later 5 isolates were recovered ~40 months after intial GI colonization. Number of SNPs for each pariwise comparision on isolates are shown. Gray highlighted boxes shown SNP defferences between the 5 later strains.
Serial passages of 4 clinical isolates.
T1 and T4 harbored ΔpmutS. Ti=Tinitial (baseline) isolates Conclusion. MMR mutations emerged in longitudinal CRKP, which conferred HM phenotypes and were associated with CAZ and other anti-CRE antibiotic resistance. mutH V76 is crucial in MMR. Long-term colonization or recurrent infections in face of antibiotic exposure might predispose CRKP strains to HM.

Presence of Antibody Dependent Cell Cytotoxicity (ADCC) Functional Antibodies that Target a Complex Gp41 Epitope Correlates with Long-term Nonprogression and ADCC is Maintained with Mutants Using Germline Heavy Chain Variable Gene Sequence of VH1-02 Gene
Sarah Baron, BA 1 ; Meghan Garrett, PhD 2 ; Mark D. Hicar, PhD 1 ; 1 University at Buffalo, Buffalo, New York; 2 University of Washington, Seattle, Washington

Session: P-56. Microbial Pathogenesis
Background. Recent data supports that improved qualitative antibody responses correlate with elite controllers (EC) of HIV. As ADCC has been associated with protection in vaccine studies, thorough exploration of antibodies that facilitate ADCC is warranted. In studies on monoclonal antibodies from long-term non-progressors (LTNPs), our laboratory has previously described highly mutated antibodies against a complex conformational epitope with contributions from both gp41 heptad repeat regions. Despite using the VH1-02 gene segment, known to contribute to some of the broadest neutralizing antibodies against HIV, members of these antibodies, termed group 76C antibodies, did not exhibit broad neutralization.
Methods. Our goal was to characterize the non-neutralizing functions of antibodies of group 76C, to assess targeting of the epitope in various clinical presentations, and to assess the development of these antibodies by comparison to their predicted common ancestor. Serum samples were obtained from HIV+ clinical groups: EC, LTNP, stable CD4 counts on therapy, and those off therapy.
Results. In antibody/serum competition assays, comparison to VRC01 which also uses VH1-02, showed that antibodies targeting the 76C group epitope were enriched in LTNPs. We then show recombinant antibodies of 76C members 6F5 and 6F11 both have robust ADCC activity, despite their sequence disparity. Sequence analysis predicted the common ancestor of this clonal group would utilize the germline non-mutated variable gene. We produced a recombinant ancestor Ab (76Canc) with a heavy chain utilizing the germline variable gene sequence paired to the 6F5 light chain. 76Canc binds HIV envelope constructs near the original group C epitope. 76Canc also shows comparable ADCC to 6F5 and 6F11 on both clade B and C constructs.
Common ancestor antibodies maintain function and these types of antibodies correlate to a non-progressive clinical state.
(A) Serum from long-term non-progressors (LTNPs) compared to serum from a group of HIV infected with lower CD4 levels as a control for viral load were used to compete against biotinylated CD4 binding site (VRC01) and 76C Gp41 conformational epitope (6F11) targeting antibodies. Serum dilutions were chosen to align means near 50%. Means with 95% confidence intervals are shown. (B) Monoclonal antibody 76Canc was created using the germline sequence of the heavy chain variable region with the CDR3 and light chain of 76C member. Antibody dependent cell cytotoxicity flow cytometric based assays were performed using gp41 proteins from clade B (MN) and clade C (ZA1197).
Conclusion. Certain antibodies present early on in infection may contribute to overall clinical course. Variable gene germline sequences that support functional activity against HIV could be targeted in vaccine regimens. Background. Multidrug resistant Acinetobacter baumannii (MDR-Ab) is a Gram-negative bacterium known for causing severe nosocomial infections, attributed in part to its formation of biofilm. Siderophore is a virulence factor known to support biofilm formation by regulating iron availability. In this study, we screened 44 isolates of MDR-Ab from our Gram-negative repository to determine the strains that phenotypically form biofilm and produce siderophore. The results were compared to Pseudomonas aeruginosa PAO1, which produces both biofilm and siderophore.

Biofilm Formation in Acinetobacter Baumannii
Methods. Isolates were grown overnight in minimal M9 medium supplemented with casamino acids and hydroxyquinones at 37°C. Bacterial cells were normalized (to OD 600=0.01) and a standard diluted 10 -3 tube was used in the study. A 96-well plate was inoculated with 100 microliters of each isolate in quadruplicates. This process was repeated in Tygon tubes with 50 microliters of each isolate in triplicates. The plate and Tygon tubes were incubated statically for 48 hours at 30°C and then stained with crystal violet. The contents were dissolved in 33% glacial acetic acid and analyzed by spectrophotometry to measure biofilm formation. Siderophore secretion was measured in supernatants with Chrome Azurol S (CAS) reagent and production was observed on CAS agar plates.
Conclusion. Many strains of MDR-Ab readily form biofilm. Overall siderophore production is lower in MDR-Ab compared to consistent production by PAO1, but this does not appear to affect MDR-Ab's ability to form biofilm. Unlike in PAO1, biofilm formation in MDR-Ab may occur independently of siderophore production. This research serves as a basis for understanding future MDR-Ab biofilm elimination in patient catheters and indwelling devices.
Disclosures. All Authors: No reported disclosures