Comparative evaluation of trimeric envelope glycoproteins derived from subtype C and B HIV-1 R5 isolates.
We previously reported that an envelope (Env) glycoprotein immunogen (o-gp140DeltaV2SF162) containing a partial deletion in the second variable loop (V2) derived from the R5-tropic HIV-1 isolate SF162 partially protected vaccinated rhesus macaques against pathogenic SHIV(SF162P4) virus. Extending our studies to subtype C isolate TV1, we have purified o-gp140DeltaV2TV1 (subtype C DeltaV2 trimer) to homogeneity, performed glycosylation analysis, and determined its ability to bind CD4, as well as a panel of well-characterized neutralizing monoclonal antibodies (mAb). In general, critical epitopes are preserved on the subtype C DeltaV2 trimer; however, we did not observe significant binding for the b12 mAb. The molecular mass of subtype C DeltaV2 trimer was found to be 450 kDa, and the hydrodynamic radius was found to be 10.87 nm. Our data suggest that subtype C DeltaV2 trimer binds to CD4 with an affinity comparable to o-gp140DeltaV2SF162 (subtype B DeltaV2 trimer). Using isothermal titration calorimetric (ITC) analysis, we demonstrated that all three CD4 binding sites (CD4-BS) in both subtype C and B trimers are exposed and accessible. However, compared to subtype B trimer, the three CD4-BS in subtype C trimer have different affinities for CD4, suggesting a cooperativity of CD4 binding in subtype C trimer but not in subtype B trimer. Negative staining electron microscopy of the subtype C DeltaV2 trimer has demonstrated that it is in fact a trimer. These results highlight the importance of studying subtype C Env, and also of developing appropriate subtype C-specific reagents that may be used for better immunological characterization of subtype C Env for developing an AIDS vaccine.
Novartis Vaccines and Diagnostics, Inc., 4560 Horton Street, Emeryville, CA 94608, USA. indresh.srivastava@novartis.com
Indresh K
IK
Srivastava
Elaine
E
Kan
Yide
Y
Sun
Victoria A
VA
Sharma
Jimna
J
Cisto
Brian
B
Burke
Ying
Y
Lian
Susan
S
Hilt
Zohar
Z
Biron
Karin
K
Hartog
Leonidas
L
Stamatatos
Ruben
R
Diaz-Avalos
R Holland
RH
Cheng
Jeffrey B
JB
Ulmer
Susan W
SW
Barnett
D000818:Animals; D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015704:CD4 Antigens / Q000378:metabolism; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D000939:Epitopes; D015967:Gene Expression Regulation, Viral; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014644:Genetic Variation; D015488:HIV Antigens; D015497:HIV-1 / Q000145:classification* / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D015704:CD4 Antigens; D000939:Epitopes; D015686:Gene Products, env; D015488:HIV Antigens