Our pseudotype particles that express RABV envelope/G-proteins are antigenically similar to those native proteins on wild-type, live RABV virus particles, which function to allow particle entry and are neutralized by anti-RABV sera. Using an antibody shown previously to detect RABV G-protein, we were able to detect these proteins in immunoblots conducted with our CVS-11 pseudotype virus. This antibody does not detect G-protein in ΔEnv virus (lacking a RABV envelope glycoprotein) or on pseudotypes bearing either the HIV envelope or VSV G-protein, although VSV and RABV are members of the same virus family, Rhabdoviridae. However, using a VSV-G monoclonal antibody, we observed the same, if not marginally less, incorporation of the envelope protein into the pseudotype. The levels of CVS-11 and VSV G-protein expression are also comparable in the transfected 293T producer cells, suggesting that the G-proteins incorporate with equal efficiency into the pseudotypes. As expected, the cleaved Gag p24 protein predominates in the mature pseudotype particle and the unspliced Gag p53 predominates in the 293T cells.
The use of replication-competent isolates of highly pathogenic viruses is strictly regulated and, in the majority of countries, requires the use of category 3 or 4 containment laboratories. Using pseudotype technology, we have constructed modified lentiviral vectors and established an assay that utilizes these replication-incompetent particles to measure RABV VNA titres accurately. This method has the benefit of allowing experiments to be undertaken in category 2 biosafety laboratories, since the pseudotype is unable to replicate and cause a productive infection. The assay also benefits from detecting VNA alone, in contrast to ELISA, but similar to FAVN, gives a more accurate picture of the protective antibodies present in the sample. Over 60 sera from various host species were evaluated for neutralizing antibodies against the CVS-11 G-protein. The results show that the pseudotype assay is 100
% specific and equally sensitive compared with the WHO/OIE-approved FAVN assay, as shown by effective differentiation between positive and negative sera and by accurately reflecting VNA titres for the positive sera.
It has been reported that human sera from recipients of the RABV HDCV can cross-neutralize EBLV-1 and EBLV-2 (Brookes et al., 2005
; Hanlon et al., 2005
). Confirmation of these observations using pseudotypes is consistent with the notion that G-protein is the target for neutralizing antibody because it is the only lyssavirus protein present in the pseudotype particle. Furthermore, our results show that antibodies in serum from vaccinated animals, specifically targeting the G-protein, cross-neutralize distinct lyssavirus genotypes to varying titres depending on viral genotype and the animal that the serum was obtained from. G-protein antibody responses in humans and cats were higher against CVS-11 infections, followed by EBLV-2 and finally EBLV-1, but additional samples need testing to confirm this observation. This result is to be expected to some extent, given that the samples were from RABV-vaccinated individuals. As expected, the rabbit serum, which was raised against EBLV-2, neutralized this genotype at higher dilutions than CVS-11 and EBLV-1.
Our cross-neutralization data for CVS-11 versus EBLV-1 or EBLV-2 support the hypothesis that the higher the degree of similarity between G-protein sequences the more comparable the neutralization titres (Brookes et al., 2005
; Hanlon et al., 2005
). The lower r2
value for EBLV-1 versus EBLV-2 does not support this hypothesis, however, as the EBLVs have a higher sequence identity to each other than to CVS-11. It is likely that this is due to all but one of the sera used in these experiments being raised only against RABV-based vaccines. In addition, the small sample size and individual variation may influence the results. This illustrates the difficulties in interpretation of antigenic differences using cross-neutralization data. A wider study using multiple representatives from each genotype, with sera raised against multiple genotypes, may assist in further quantifying antigenic differences between lyssaviruses.
The cross-neutralization of lentiviral pseudotypes bearing lyssavirus envelopes will provide the opportunity to analyse neutralizing epitopes in G-proteins quantitatively and qualitatively. The method described here can be extended to other lyssaviruses in phylogroups 1 and 2. Because cDNA cloned into plasmids is used to generate the pseudotype, site-specific mutagenesis can be exploited to elucidate which domains are crucial for G-protein function in effecting virion attachment and entry, and which neutralizing epitopes determine strain-specific and group-specific responses to sera and to monoclonal antibodies.
For the adoption of neutralization assays, using either replication-competent or pseudotype viruses, there are some issues that need to be addressed before the assay can be employed for routine serological surveillance. In both systems, there will be free or secreted envelope protein in the virus preparations that can adsorb antibody from the supernatant and thus skew neutralization tests. It is therefore important to standardize virus input in order to obtain the most sensitive and reliable results. This can be achieved by determining the concentration of virus at which a known negative serum becomes positive, and by using a concentration of virus slightly greater than that routinely used by rabies reference laboratories as part of their assay validation process. Alternatively, where a baseline positive control is available, for example the OIE reference standard for RABV neutralization assays, the smallest amount of virus that is needed to achieve an IC100 at the lowest serum dilution could be determined. This will allow for the greatest range of serum dilutions to be tested, while all negative samples will provide a ‘failed’ result. For our assay, this result was achieved using a virus input between 10×TCID50 (1×105 RLU) and 100×TCID50 (1×106 RLU). Therefore, we would recommend 10–100×TCID50 or 1×105–1×106 RLU virus input for neutralization assays using the CVS-11 pseudotype. While background readings for cells alone or virus alone were 50–200 RLU, luciferase carry-over for ΔEnv samples and those pseudotypes that produced a negative GFP result was up to 1×104 RLU, depending on the cell line used (with 50 μl pseudotype input). Therefore, the ΔEnv value should be considered the cut-off for a positive viral titre when using large volumes of pseudotype. However, as only 0.1–0.5 μl of pseudotype virus is normally used, the ‘virus alone’ luciferase reading is generally larger than that which would be observed with ΔEnv and is therefore used as the background value.
It was encouraging to observe that BHK cells, which are routinely used in live virus CVS-11 FAVN tests, were highly permissive for our CVS-11 pseudotype. Conversely, N2A cells that were readily infected by live lyssaviruses were not permissive for the CVS-11 pseudotype with HIV core. In contrast, MLV-based pseudotypes, made using different reporter constructs, were able to infect N2A cells, suggesting a post-entry restriction imposed selectively on the lentiviral reporter gene in the vector. This could be due to a specific restriction of the pCSGW and pCSFLW promoter in this cell line, because N2A cells were also almost completely refractory to infection by VSV, EBLV-1 and EBLV-2 pseudotypes produced with the same HIV reporter plasmids (data not shown). Alternatively, the lentiviral core may be subject to a post-penetration restriction in N2A cells, such as TRIM5α
The fatality rate of RABV infections in humans who have not received either pre- or post-exposure prophylaxis is very high, nearing 100
%. Compared with the diseases caused by other highly pathogenic viruses, such as Ebola virus haemorrhagic fever (80
% fatality) and H5N1 influenza (61
%), the ongoing research and surveillance for RABV is limited, supporting the suggestion that rabies is a ‘neglected disease’, especially in rabies-endemic countries in Africa and Asia (WHO, 2006
; Fooks, 2005
). There is, therefore, a need to readdress this balance by increasing the current level of surveillance for RABV in countries where the infection causes high levels of mortality. This would also help to limit misdiagnosis and consequent underreporting of RABV infection (Mallewa et al., 2007
). The main assay currently being used (FAVN) provides good sensitivity and specificity, but requires handling of live RABV. Alternative assays for the detection of RABV antibodies, such as ELISA (Cliquet et al., 2004
) and rapid fluorescent focus inhibition test-GFP (Khawplod et al., 2005
), have been designed; however, the former does not differentiate between neutralizing and non-neutralizing antibodies and the latter still requires high containment-level facilities.
The use of pseudotypes for the detection of VNAs removes the need to use live viruses and provides both high sensitivity and high specificity for the detection of neutralizing antibodies. Incorporation of GFP or luciferase as a reporter gene makes this assay applicable to many laboratories involved in RABV surveillance, and a good candidate for high throughput screening. For laboratories lacking fluorescence or luciferase detection, a β-galactosidase reporter could be used. It is also amenable to testing for the presence of antibodies in small volumes of sera (microassay) such as may be obtained from bats. In conclusion, this report shows that it is possible to analyse cross-neutralizing antibody responses against different lyssavirus genotypes using lentiviral pseudotypes. This approach could greatly improve the surveillance of new and emerging lyssaviruses and evaluation of the protection that current vaccines offer against these pathogens.