Passage of dengue virus type 4 vaccine candidates in fetal rhesus lung cells selects heparin-sensitive variants that result in loss of infectivity and immunogenicity in rhesus macaques

Abstract

Three dengue virus type 4 (DENV-4) vaccine candidates containing deletions in the 3' noncoding region were prepared by passage in DBS-FRhL-2 (FRhL) cells. Unexpectedly, these vaccine candidates and parental DENV-4 similarly passaged in the same cells failed to elicit either viremia or a virus-neutralizing antibody response. Consensus sequence analysis revealed that each of the three viruses, as well as the parental DENV-4 when passaged in FRhL cells, rapidly acquired a single Glu327-Gly substitution in domain III (DIII) of the envelope protein (E). These variants appear to have accumulated in response to growth adaptation to FRhL cells as shown by growth analysis, and the mutation was not detected in the virus following passage in C6/36 cells, primary African green monkey kidney cells, or Vero cells. The Glu327-Gly substitution was predicted by molecular modeling to increase the net positive charge on the surface of E. The Glu(327)-Gly variant of the full-length DENV-4 selected after three passages in FRhL cells showed increased affinity for heparan sulfate compared to the unpassaged DENV-4, as measured by heparin binding and infectivity inhibition assays. Evidence indicates that the Glu327-Gly mutation in DIII of the DENV-4 E protein was responsible for reduced infectivity and immunogenicity in rhesus monkeys. Our results point out the importance of cell substrates for vaccine preparation since the virus may change during passages in certain cells through adaptive selection, and such mutations may affect cell tropism, virulence, and vaccine efficacy.

FIG. 1.

Electropherograms from the consensus sequence analysis of full-length DENV-4 and its derived deletion mutants. The parental virus constructs recovered from C6/36 cells were passaged in FRhL cells at levels 1, 2, and 3. Nucleotides 1917, 1918, and 1919 in bold constitute the codon for amino acid position 606 of the polypeptide (or position 327 in the E protein). Nucleotide change A₁₉₁₈→G is shown in red.

FIG. 2.

Immunofocus assay showing the plaque size morphology of parental DENV-4 and its FRhL cell-selected variant on Vero cell monolayers. (A) DENV-4 strain 814669 prior to passage in FRhL cells. (B) Passage 1. (C) Passage 2. (D) Passage 3.

FIG. 3.

Increased replication and infectivity of DENV-4 variants in FRhL cells. (A) Growth curves of parental DENV-4 strain 814669 and its derived variants DENV-4(Gly₃₂₇) and DENV-4(Ala₃₂₇) in FRhL cells infected at an MOI of 0.01. Virus titers in the culture fluid were determined by FFA in Vero cells. (B) Quantitative analysis of viral RNA in the culture fluid by qRT-PCR. The number of viral RNA copies in GEq/ml was calculated. (C) The ratio of infectivity to GEq (FFU/GEq) of parental DENV-4 and its variants 4 days after infection. p.i. postinfection.

FIG. 4.

A positively charged surface patch in DIII of two DENV-4 variants increases its binding to heparin. (A) Molecular modeling and surface mapping of the electrostatic field of DENV-4 E DIII in the indicated viruses. Blue and red denote positive and negative charges, respectively. Field charge distribution was calculated using Adaptive Poisson-Boltzmann Solver software. Amino acid position 327 is shown by a yellow arrow. Both parental and variant structures were modeled into the nuclear magnetic resonance-derived solution structure of DIII of the DENV-4 E (Protein Data Bank code 2H0P) (54) using the software VMD, version 1.8.6. (B) Heparin inhibition of full-length DENV-4 infectivity in Vero cells infected with the indicated viruses. Each sample point was performed with 1 × 10⁴ FFU of the virus in duplicate, mixed with heparin at concentrations ranging from 0.2 to 200 μg/ml. (C) Heparin binding assay of full-length DENV-4. Virus binding to heparin was determined through virus removal by heparin-Sepharose beads. Student's t test was used to compare values between groups, and a P value ≤0.05 was considered statistically significant.