Short Communication: Evaluating the Level of Expressed HIV Type 1 gp120 and gag Proteins in the vCP1521 Vector by Two Immunoplaque Methods

O ver the past few years, live viral canarypox vectors have been used in several vaccine trials. ^{1 –5} The ALVAC vector, a modified form of canarypox, demonstrated several advantages over other conventional live viral vectors. ^{6 –9} However, one of the concerns with recombinant ALVAC vectors is the occurrence of mutations and deletions within genes in the recombinant construct, which may affect the stable expression of proteins. ¹⁰ This point was also discussed in a WHO meeting in 2003, where the quality, efficacy, safety, and regulatory requirements of live viral vaccines including ALVAC vectors were reviewed and documented. ¹¹ The committee recommended performing reliable potency assays for monitoring the consistency of gene expression in viral vectors during manufacturing. The regulatory requirements for the expression of genetic insert products in poxvirus vectors were further documented in section 5.14, “Gene Transfer Medicinal Products for Human Use,” in the European Pharmacopeia 7.0. ¹⁰

In the RV144 study, the combination of our ALVAC-HIV vector vCP1521 (prime) and AIDSVAX B/E gp120 (boost) has been used. ^{12 –15} The results from the RV144 study as a “proof of concept” provided support to proceed with additional HIV vaccine trials using the vCP1521 construct. Therefore, two lots of vCP1521 viral vectors (ALVAC-HIV-00A1 and ALVAC-HIV-00A2) expressing HIV-1 gp120 and gag proteins have been manufactured. The ability of each lot of vCP1521 to express HIV antigens was characterized by two modified immunoplaque approaches (dish and plaque lift methods). An empty ALVAC vector also has been used as a control in our assays.

Primary chick embryo fibroblast (CEF) cells (Charles River SPAFAS), the cell substrate of choice for ALVAC vectors, have been used in both methods. Briefly, a day prior to use, CEF cells were counted and suspended at a concentration of 2.5×10⁶ cells per ml in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (FBS) (Gibco), 1% sodium pyruvate (Gibco), and 1% penicillin/streptomycin (Gibco). Each 60-mm plate (Falcon) was seeded with 5 ml of suspended CEF cells and incubated overnight at 36°C±1°C, 5%±2% CO₂. The next day, plates were inspected under the microscope to ensure the cell monolayer was over 80% confluent, healthy, and free of contamination. Thereafter, the medium was removed from the plates and the confluent cells were infected with the appropriate amounts of ALVAC-HIV-00A1, ALVAC-HIV-00A2, or ALVAC-C viral vectors. The plates were incubated at 36°C±1°C, 5%±2% CO₂ for 1 h and gently rocked every 15 min to allow adsorption of the ALVAC viral constructs. The vector inocul were aspirated from the plates and the CEF monolayers were overlaid with 2XMEM (Gibco)+10% FBS and 1.2% agarose (Sigma) at 36°C±1°C, 5%±2% CO₂ for 6 days.

To perform the dish immunoplaque method, agarose was removed 6 days postinfection, the cell monolayer was rinsed with phosphate-buffered saline (PBS), and methanol was added. After fixation, CEF cells were washed with PBS supplemented with 0.1% Saponin (PBS-S) (Sigma) and each plate was stained with either 1:250 diluted mouse monoclonal HIV gp120 antibody (obtained from R. Kennedy, Texas) or 1:200 diluted mouse monoclonal HIV1-gag antibody (Dupont NEN research) for 1 h at 36°C±1°C, 5%±2% CO₂. After incubation, the primary antibodies were removed and plates were washed with PBS-S. After washing, horseradish peroxidase (HRP) conjugated rat anti-mouse antibody (1:5000) (Jackson Immunoresearch) was added to the plates. After a 30 min incubation at 36°C±1°C, 5%±2% CO₂, plates were washed with PBS-S and peroxidase substrate (Fisher Scientific, metal enhanced DAB kit) was added to each plate for 15 min. The reaction was stopped by adding water to each plate and the number of plaques was counted.

Next, the developed plates were stained with 1:100 diluted APOX (an in-house antibody for staining ALVAC constructs) to measure the total numbers of plaques. The plates were washed, stained with the HRP-conjugated antibody, and developed as described above. The ratio between the plaques expressing either gp120 or gag protein to the total number of ALVAC plaques present determines the percentage of vCP1521 vector expressing the desired proteins. While this method was able to detect the expression of gp120 and gag proteins, it was not able to determine if each individual plaque expressed both gp120 and gag proteins.

A complementary approach to the dish method is the plaque lift method. In this approach, CEF cells were cultured and infected as detailed above. Six days postinfection, agarose was removed with a spatula and a labeled nylon membrane (VWR, NYL-66) was placed on the top of the cell monolayer. A filter paper (VWR) soaked in sterile 50 mM Tris and 0.15 M NaCl (pH 7.6) was placed on top of the nylon membrane and gentle pressure was applied. Next, the nylon membrane was peeled off with the cell monolayer attached. A new labeled replica nylon membrane soaked in sterile 50 mM Tris and 0.15 M NaCl was placed on the top of the original nylon membrane for 5 min. The replica nylon membrane was carefully pulled apart from the original nylon membrane and stored between dry filter papers. The replica nylon membranes were air dried at room temperature for 2 h and they were stored at −30°C to −20°C or blocked with the blocking buffer for 1 h at room temperature. The blocking buffer was made by dissolving 25 g skim milk powder (BD Difco) in 500 ml Tris-buffered saline (TBS) buffer (1 mM Tris-HCl, pH 8.0, 150 mM NaCl).

After incubation, blocking buffer was removed and each labeled replica nylon membrane was incubated with either 1:250 diluted mouse monoclonal HIV gp120 antibody, 1:200 diluted mouse monoclonal HIV gag antibody, or 1:100 diluted APOX antibody for 1 h at room temperature. After incubation, the replica nylon membranes were washed two times with TBS-T (TBS containing 0.1% Tween-20) and the replica filter membranes were incubated with 1:5000 diluted HRP-conjugated rat anti-mouse antibody (Jackson Immunoresearch) for 30 min at room temperature. After incubation, membranes were washed twice with TBS-T followed by one wash with 1×TBS alone, and the plaques were developed with a chemiluminescence reagent (Perkin Elmer). In this study, the transfer from the original nylon membrane was repeated three times to have enough replica nylon membranes for each of gp120, gag, or APOX antibodies. As an example, two replica nylon membranes that were stained with APOX, or gp120 specific antibodies are shown in Fig. 1.

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FIG. 1.

Image of replica plaque lifts stained with either APOX (ALVAC-specific), P24 (gag-specific), or K3A (gp120-specific) antibodies. Gray arrows indicate the location of a plaque stained with the parental antibodies (APOX), illustrating expression of gp120 but lack of gag protein expression for this specific plaque. Black arrow identifies a plaque that expresses both proteins (gp120 and gag). This example supports the suitability of this technique to directly identify and potentially isolate plaques for further analysis and evaluation for the stability of viral constructs upon passage in tissue culture.

The plaque lift method was also able to demonstrate a specific result for monitoring the consistency of gp120 and gag protein expression by the vCP1521 vector lots. Additionally, this method was able to determine if each plaque of vCP1521 vector expressed either gag, gp120, or both genes. The plaque lift method is more suitable for viral vector studies, where multiple genes are inserted into one vector but may not express all the proteins. In such studies, the plaque of interest could be isolated easily from the nylon membrane and cultured in appropriate media. Afterward, the virus grown can be isolated, sequenced, and analyzed in order to determine if the construct was not stable. Therefore, the plaque lift method appears to be a more suitable approach than the dish method for this gene expression study.

Overall, a comparison between the data obtained by the dish and plaque lift methods in two lots of vCP1521 demonstrated consistency between the results. The expression of gp120 and gag proteins by the dish method in the ALVAC-HIV-00A1 vector was 99.3% present in the plaques produced. These results were similar to the plaque lift method, which showed 99.7% and 99.0% expression of proteins for gp120 and gag, respectively, in the plaques produced. The percent expression of gp120 and gag as determined by either the dish or plaque lift method are shown in Table 1. Antibodies used for each specific gene product are noted in parentheses.

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Table 1.

Percent Expression of gp120 and gag Proteins by the Dish or Plaque Lift Method

	Dish method		Plaque lift method
vCP1521 lot number	gp120 (K3A)	gag (P24)	gp120 (K3A)	gag (P24)
ALVAC-HIV-00A1	99.3%	99.3%	99.7%	99.0%
ALVAC-HIV-000A2	99.5%	99.5%	99.8%	99.7%

To evaluate the coexpression of HIV-1 antigens in the constructs, other methods such as FACS analysis of various infected cell lines with antibodies against ALVAC vector and HIV-1 proteins have also been tested. The FACS analysis showed the capacity of each construct to express HIV-1 antigens, confirming the results of plaque methods (data not shown).

The evaluation of potency in human ALVAC vaccine studies is one of the critical regulatory requirements. The characterization tests described highlight the ability of both methods for monitoring the expression of inserted genes in manufactured vCP1521 recombinant ALVAC vector, complying with the regulatory guidelines. In addition, these two methodologies could be easily extended as characterization tests for other viruses and viral vectors.