Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2019 Jun 14;7(2):51.
doi: 10.3390/vaccines7020051.

An Influenza Virus Hemagglutinin-Based Vaccine Platform Enables the Generation of Epitope Specific Human Cytomegalovirus Antibodies

Mohammad Amin Behzadi  1 , Kathryn R Stein  2 , Maria Carolina Bermúdez-González  3   4 , Viviana Simon  5   6   7 , Raffael Nachbagauer  8 , Domenico Tortorella  9
Affiliations

An Influenza Virus Hemagglutinin-Based Vaccine Platform Enables the Generation of Epitope Specific Human Cytomegalovirus Antibodies

Mohammad Amin Behzadi et al. Vaccines (Basel). .

Abstract

Human cytomegalovirus (CMV) is a highly prevalent pathogen with ~60%-90% seropositivity in adults. CMV can contribute to organ rejection in transplant recipients and is a major cause of birth defects in newborns. Currently, there are no approved vaccines against CMV. The epitope of a CMV neutralizing monoclonal antibody against a conserved region of the envelope protein gH provided the basis for a new CMV vaccine design. We exploited the influenza A virus as a vaccine platform due to the highly immunogenic head domain of its hemagglutinin envelope protein. Influenza A variants were engineered by reverse genetics to express the epitope of an anti-CMV gH neutralizing antibody that recognizes native gH into the hemagglutinin antigenic Sa site. We determined that the recombinant influenza variants expressing 7, 10, or 13 residues of the anti-gH neutralizing antibody epitope were recognized and neutralized by the anti-gH antibody 10C10. Mice vaccinated with the influenza/CMV chimeric viruses induced CMV-specific antibodies that recognized the native gH protein and inhibited virus infection. In fact, the influenza variants expressing 7-13 gH residues neutralized a CMV infection at ~60% following two immunizations with variants expressing the 13 residue gH peptide produced the highest levels of neutralization. Collectively, our study demonstrates that a variant influenza virus inserted with a gH peptide can generate a humoral response that limits a CMV infection.

Keywords: gH envelope protein; hemagglutinin; human cytomegalovirus; humoral immunity; influenza virus; neutralization; vaccine.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Model of the chimeric influenza hemagglutinin (HA)/CMV gH molecules. (A) Predicted structure of the CMV gH protein. Blue color indicates the mAb 10C10 recognition epitope. (B) Predicted structure of influenza A virus (IAV) PR8 HA trimer (PDB3LZG) (top view and side view) with the classically defined antigenic site Sa colored in pink. The AA numbering is based on Burke Reference Sequence Alignment for PR8 [43]. (C) Predicted structures of chimera HAs containing CMV gH epitope (7AA, 10AA, or 13AA, in blue) within Sa antigenic site (in pink). Modeling performed with PyMOL (The PyMOL Molecular Graphics System, Version 2.0.1, Schrödinger, LLC) [44]. Amino acids in gray are unchanged. (D) Amino acid sequences of the Sa antigenic site (in pink) of chimera IAVs (A/Puerto Rico/8/1934(H1N1)) with CMV gH epitopes (7AA, 10AA, or 13AA, in blue).
Figure 2
Figure 2
The gH peptide within the HA/gH chimeric viruses is conformationally similar to the native CMV gH protein. (A) Immunofluorescence staining of HEK293T cells transfected with plasmids encoding the HA/gH chimeras (HA/gH-7AA, HA/gH-10AA, and HA/gH-13AA), wild-type (wt) HA, and the full CMV gH protein were probed using anti-influenza A monoclonal antibody (KB2) and anti-CMV gH monoclonal antibody (10C10). Empty vector was utilized as a negative control. (B) MDCK cells infected with the chimeric HA/gH and wt-influenza viruses (MOI 0.25) were probed at 48 hpi with 10C10 and anti-influenza A polyclonal mouse serum (α-IAV sera). (C) Growth curves in MDCK cells of the chimera HA/gH viruses (HA/gH-7AA, HA/gH-10AA and HA/gH13AA) and of the wt-influenza virus (wt virus).
Figure 3
Figure 3
An anti-gH antibody neutralizes the chimeric influenza/CMV viruses. (A) Minimal hemagglutination inhibition assays was performed with increasing dilutions (two-fold dilutions starting from 100 µg/mL) of the anti-cytomegalovirus (CMV) gH monoclonal antibody (mAb) (10C10) against wild type influenza virus (wt virus) and chimera HA/gH influenza viruses (HA/gH-7AA, HA/gH-10AA and HA/gH-13AA). The accumulation of chicken red blood cells at the bottom of well indicates the lack of inhibition. (B) Wild type influenza virus (wt virus) and chimera HA/gH influenza viruses (HA/gH-7AA, HA/gH-10AA, and HA/gH-13AA) were subjected to influenza neutralization assay (in triplicate) with increasing concentrations (two-fold dilutions starting from 100 µg/mL) of mAb 10C10, PR8 head specific mAb (PY102), and HA stalk specific mAbs (KB2 and CR9114) against wt virus and chimera HA/gH viruses. The virus from infected cells was titered using a classical hemagglutination assay and plotted based on the minimal mAb concentration that neutralizes 100 TCID50 of influenza virus.
Figure 4
Figure 4
Chimeric influenza/CMV viruses generate a humoral response against CMV gH. (A) The immunization (syringe) and blood collection strategy for groups of five mice immunized with either chimera HA/gH viruses HA/gH-7AA (7AA), HA/gH-10AA (10AA), and HA/gH-13AA (13AA) or the wild type IAV virus (wt) lacking CMV components is depicted. Blood collection occurred prior to immunization. (B) Pooled sera from each immunization group was analyzed for specificity to cells expressing the CMV gH/gL dimer by measuring immunoglobulin binding followed by anti-mouse Ig-conjugated to Alexa647 (αmouse-IgAlexa647) with flow cytometry. Controls included anti-gH (10C10) and anti-gB (2F4) (2 and 0.4 µg/mL) mAbs as well as cells probed with only αmouse-IgAlexa647 (Mock). (C) Serum from individual mice at 84 days post prime (dpp) was subjected to flow cytometry analysis of CMV gH/gL-expressing cells. Anti-CMV gH mAb were utilized as a positive and negative control, respectively. Mock represents cells probed with αmouse-IgAlexa647only and control cells refer to non-gH/gL expressing cells. The mean fluorescence intensity (MFI) was determined using FlowJo Software and the data points were from technical replicates with s.d. depicted. * p < 0.05, ** p < 0.01, **** p < 0.0001 (ANOVA).
Figure 5
Figure 5
Chimera influenza/CMV viruses generate a CMV inhibition antibody response. (A) Pooled serum from each immunization group was analyzed in a CMV inhibition assay with the infection of AD169BADrUL131 (MOI: 0.5) pre-incubated with serum (1:100) in ARPE-19 cells. Cell infection was analyzed at 24 h post-infection using the Celigo fluorescence cytometer. Anti-CMV gH monoclonal antibody (mAb) (10C10) (2 µg/mL) and PR8 head specific mAb (PY102) (2 µg/mL) were utilized as positive and negative controls, respectively. (B) Serum from individual mice at 84 days post prime (dpp) was subjected to a CMV inhibition assay with infection of AD169BADrUL131 (MOI: 0.5) pre-incubated (1:100 and 1:500) with sera at specified dilutions in ARPE-19 cells. Pre-incubation with serum from a mouse immunized with the whole CMV (TB40/E) virus (M*) (1:500 and 1:1500), normal mouse serum (NMS) (1:100 and 1:500), and mAbs 10C10 and PY102 (2 µg/mL) were utilized as controls. The % inhibition was determined using NMS as 0% inhibition and no infected cells as 100% inhibition. Experiments were performed in technical replicates with the s.d. being depicted. * p < 0.05, **** p < 0.0001 (ANOVA).
See this image and copyright information in PMC

Similar articles

See all similar articles

References

    1. Mocarski E.S. Human Herpesviruses: Biology, Therapy, and Immunoprophylaxis. Cambridge University Press; Cambridge, UK: 2007. Betaherpes viral genes and their functions. - PubMed
    1. Damato E.G., Winnen C.W. Cytomegalovirus Infection: Perinatal Implications. J. Obstet. Gynecol. Neonatal Nurs. 2002;31:86–92. doi: 10.1111/j.1552-6909.2002.tb00026.x. - DOI - PubMed
    1. Cannon M.J., Pellett P.E. Risk of Congenital Cytomegalovirus Infection. Clin. Infect. Dis. 2005;40:1701–1702. doi: 10.1086/430172. - DOI - PubMed
    1. Cheeran M.C.J., Lokensgard J.R., Schleiss M.R. Neuropathogenesis of congenital cytomegalovirus infection: Disease mechanisms and prospects for intervention. Clin. Microbiol. Rev. 2009;22:99–126. doi: 10.1128/CMR.00023-08. - DOI - PMC - PubMed
    1. Dove A. A Long Shot on Cytomegalovirus. [(accessed on 14 June 2019)]; Available online: https://www.the-scientist.com/uncategorized/a-long-shot-on-cytomegalovir....

LinkOut - more resources