Patent Application
20080085288
The viremia and the immunogenicity were tested in a monkey model. The viremia, in particular, was identified as one of the factors associated with the virulence and the severity of the disease in man and therefore constitutes an important parameter to be taken into consideration. The immunogenicity is, for its part, a key parameter in the context of the evaluation of the protection conferred.
1.1 Materials and Methods:
The experiments in monkeys were carried out according to the European Directives relating to animal experimentation. The immunizations were carried out in cynomolgus monkeys (Macaca fascicularis) originating from Mauritania. The monkeys were placed in quarantine for six weeks before immunization.
The monkeys were immunized subcutaneously in the arms with 0.5 ml of vaccinal composition. After a light anesthesia with ketamine (Imalgene, Merial), blood was collected by puncture from the inguinal or saphenous veins. At day 0 and 28 following each immunization, 5 ml of blood were sampled in order to evaluate the antibody responses, while, between days 2 and 10, 1 ml of blood was sampled in order to evaluate the viremia. The blood was collected on ice and stored on ice until serum separation. To do this, the blood was centrifuged for 20 minutes at 4° C. and the serum collected was stored at −80° C. until the time of the tests.
Measurement of Viremia
The post-vaccinal viremias were monitored by quantitative real-time RP-PCR (qRT-PCR). Two sets of primers and of probes located in the NS5 gene of the DEN1 and DEN2 strains were used to quantify the VDV-1 RNA and VDV-2 RNA, respectively. A third set of 2 primers and of 1 probe located in the NS5 gene of the YF virus was used to quantify the CYD RNA. Finally, 4 sets of primers and of probes specific for the various CYD serotypes, located at the junction of the E (DEN)/NS1 (YF) genes were used to identify the serotype in the samples positive for the YF NS5 RNA (see also table I). 7 plasmids containing, under the control of the T7 promoter, the region targeted by each PCR were transcribed in vitro so as to generate a series of synthetic RNAs which were included as an internal reference in each RT-PCR assay. These synthetic RNAs were assayed by spectrophotometry, and the amount of RNA obtained was converted to number of RNA copies and expressed as GEQ (genomic equivalents).
0.140 ml of monkey serum was extracted using the Macherey Nagel “Nucleospin 96 virus™” RNA extraction kit, according to the manufacturer's instructions, and then the purified RNA was eluted with 0.140 ml (0.090 ml, then 0.05 ml) of RNase-free water. In order to avoid repeated freezing/thawing cycles, a first quantification was carried out immediately after the extraction, on 5 μl of said RNA preparation. The remaining volume was frozen at 70° C.
The reaction mixtures contained, in addition to the components of the “Qiagen Qauntitect™ probes” RT-PCR quantification kit (Qiagen), 10 picomol of each primers, 4 picomol of each probe and 5 μl of RNA, in the total volume of 25 μl. In the case of the RNAs to be tested, 5 μl of the purified preparation were directly introduced into the reaction mixture, without any prior dilution step. The synthetic RNAs were diluted to 1/10 in RNAse-free water, and 7 dilutions containing approximately 10 to 106 GEQ in 5 μl were quantified in parallel in order to generate the standard curve.
The quantification reactions were carried out on the Applied Biosystem ABIPrism 700™ device, using the following program: 50° C./30 min, 95° C./15 min, then 40 cycles of 95° C./15 sec-60° C./60 sec.
The limit of quantification of the viral RNA in this test is from 2.9 to 3.3 log10GEQ/ml (800 to 2000 GEQ/ml; 4 to 10 GEQ/reaction), according to the PCR targets (standard deviation: +/−0.3 log10).
The correlation between the infectious titer and the viral RNA quantification was established in parallel to the assays, by analysis of 0.140 ml of negative monkey serum samples (DO) to which a known amount of infectious particles of the viruses which were used for the immunization (CYD or VDV) were added. Said control sera were prepared at two dilutions containing approximately 1 PFU and approximately 100 PFU in 5 μl (2.3 and 4.3 log10 PFU/ml, respectively).
In the tests used in the examples, the correlation between GEQ and PFU is the following:GEQ/PFU ratio of 2.7 log10 (i.e.: 1 PFU=500 GEQ) for the sera positive for YF or CYDs. GEQ/PFU ratio of 2.5 log10 (i.e.: 1 PFU=320 GEQ) for the sera positive for VDVL or VDV2.
The quantification limits being <3.3 log10GEQ/ml (i.e.: <4 PFU/ml) for qRT-PCR YF and CYDs and <2.9 log10GEQ/ml (i.e.: <2.5 PFU/ml) for qRT-PCR VDV1 and VDV2.
The primers and probes used are given in table 1 below, in which are listed, in order, for each assay, the sense and antisense primers and the probe.
Measurement of Neutralizing Antibodies (Seroneutralization Test) (SN50)
Conventionally, the dengue antibody measurement is established using the PRNT50 (50% PFU number reduction neutralization test). Since this test is laborious and uses up a lot of material, we developed the SN50 test, based on 50% reduction in the number of units measured in a CClD50 test.
In a 96-well plate, 0.120 ml of each decomplemented serum is added to 0.480 ml of diluent (ISCOVE 4% FCS) per well. 6-fold serial dilutions are prepared by transfer of 0.150 ml of serum into 0.450 ml of diluent. 450 μl of virtual dilution at 2.7 log10 CCID50/ml are added to each well so as to obtain 25 CCID50/well. The plate is incubated at 37° C. for 1 hour. 0.1 ml of each dilution is then distributed into 6 wells of a 96-well plate into which VERO cells had been seeded 3 days before the beginning of the experiment at a density of 8000 cells/well, in 0.1 ml of ISCOVE medium containing 4% FCS. After incubation at 37° C. for 6 days, in the presence of 5% CO2, the cells are fixed with an ethanol/acetone (70/30) mixture at 4° C. for 15 minutes, and then washed 3 times in PBS and incubated for 1 h at 37° C. in the presence of 0.05 ml of a 1/2000 dilution of an anti-flavivirus monoclonal antibody (mAb 4G2). The plates are then washed twice and incubated for 1 h at 37° C. in the presence of 0.05 ml of a 1/1000 dilution of an alkaline phosphatase-conjugated anti-mouse IgG. The lysis plaques are visualized by adding 0.05 ml of a colored substrate: BCIP/NBT. The neutralizing antibody titers are calculated using the Karber formula as defined below:
log10SN50=d+f/N(X+N/2),
in which:
d represents the dilution resulting in 100% neutralization (i.e. 6 negative replicates, i.e. replicates exhibiting no sign of infection)
f: represents the dilution factor in log 10 (e.g. dilution factor of 1:4, f=0.6)
The limit of viral detection is 10 SN50 (i.e. 1.0 log10SN50).
The viral strains which were used for the neutralization are the DEN1 16007, DEN2 16681, DEN3 16562 or DEN4 1036 strains. For the controls, the initial viral dilutions were re-titrated. The correlation between the neutralizing titer measured in the SN50 test and the neutralizing titer measured conventionally in the PRNT50 test is: log10PRNT50=log10SN50+0.2
The mean titer (GMT) is established by calculating the geometric mean of the titers expressed in linear value; the samples for which the titer is less than the detection threshold are, by convention, assigned a value equal to half this threshold.
1.2 Evaluation of the Sequential Immunizations
2 groups of 4 monkeys of equivalent age and weight were immunized (see table 2).
The immunization was carried out subcutaneously in the arm, with a 23G1 needle, at a dose of 105 CCID50 for each serotype for the CYD DEN 1 to 4 vaccines.
The immunogenicity results obtained after one immunization (D28) and two immunizations (D86) are given in table 3.
The viremia results are given in table 4.
Briefly, the results can be summarized as follows:
The examples therefore show that the method of immunization according to the present invention improves the immunogenicity of the vaccinal dengue viruses without impairing the safety of the latter.
The viremia and the immunogenicity were tested in the monkey model as in example 1. In the present example, the bivalent compositions tested contain, respectively, the most immunogenic vaccinal viruses (CYD-1,4) and the least immunogenic vaccinal viruses (CYD-2,3).
2.1 Materials and Methods: Identical to Example 1
2.2 Evaluation of the Simultaneous Immunizations
2 groups of 4 monkeys of equivalent age and weight were immunized (see table 5).
The immunization was carried out as described in example 1.
The immunogenicity results obtained after one immunization (D28) and two immunizations (D86) are given in table 6.
The viremia results are similar to those obtained in example 1, showing a viremia induced by serotype 4 and no significant differences between the two groups.
The results support those obtained in example 1 and can be summarized as follows:
The two examples above therefore show that the method of immunization according to the present invention improves the immunogenicity of the vaccinal dengue viruses without impairing the safety of the latter as evaluated by measuring the viremia.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR 06 08660 | Oct 2006 | FR | national |
This application claims the benefit of priority of U.S. provisional application 60/867,312, filed Nov. 27, 2006.
| Number | Date | Country | |
|---|---|---|---|
| 60867312 | Nov 2006 | US |
