Patent Application
20080038293
The following example sets forth a preferred embodiment of the present invention. It is to be understood, however, that this example is provided by way of illustration and nothing therein should be taken as a limitation upon the overall scope of the invention.
This example provides data as to the efficacy of DNA vaccines comprising various regions of the PRRSV genome. The example began with 40 PRRSV-negative mixed sex pigs from Spring Prairie Colony, Hawley, Minn. 56549. The pigs were 3-4 weeks of age at the initiation of the study. Throughout the study, the pigs were provided food sufficient for the size, age and condition of the animals. Water was provided ad libitum.
To generate PRRSV DNA vaccines, nineteen cDNA clones were generated from the PRRS virus. Thirteen cDNA clones were generated that sequentially represent the open reading frame (ORF) 1a/1b region of the pRRSV genome. Clone A utilizes the authentic ORF1a ATG start codon. The remaining ORF1a/1b clones B through M had an ATG start codon added to their respective 5′ ends. All of the above clones were respectively cloned into the DNA expression vector pVC1650. The pVC 1650 vector contains the immediate early cytomegalovirus promoter and intron A to direct the expression of the various PRRSV ORFs in eukaryotic cells. The six additional cDNA clones represented the PRRSV structural protein ORFs 2, 3, 4, 5, 6 and 7. The ORF 2, 3, 4, 5, and 6 clones were also respectively cloned into the DNA expression vector pVC1650 described above. The ORF 7 gene was cloned into a similar expression vector designated WRG7020. The WRG7020 vector also contains the immediate early cytomegalovirus promoter and intron A to direct the expression of PRRSV ORF 7 in eukaryotic cells.
Two sets of vaccines were created, designated “A1-19” and “T1-19”. For the A1-19 vaccines, the aforementioned clones were cloned into the Valentis, Inc. pVC1650 expression plasmid. Each plasmid construct was separately formulated with aluminum phosphate (Adju-Phos®) (Ulmer et al., Enhancement of DNA Vaccine Potency Using Conventional Aluminum Adjuvants; 18 Vaccine, 18-28 (1999) to yield 250 μg of respective ORF clone with 1000 μg calculated aluminum in a 1 ml dose. The final vaccine consisted of a separate 1 ml 1M dose of each formulated ORF clone. For the T1-19 vaccines, the aforementioned clones were cloned into the Valentis, Inc. pVC1650 expression plasmid. Each plasmid construct was separately formulated with TGV200 PINC-polymer to yield 250 μg of respective ORF clone in a 1 ml dose. The final vaccine consisted of a separate 1 ml 1M dose of each formulated ORF clone.
To generate a control, DNA vaccines consisting of cDNA clones of ORF PCV2 and the HA gene of SIV were created. Both ORFs were separately cloned into the Valentis, Inc. pVC1650 expression plasmid. Both the PCV2 ORF2 and the SIV HA gene plasmid constructs were separately formulated with aluminum phosphate (Adju-Phos®) to yield 250 μg of the respective ORF clone with 1000 μg calculated aluminum in a 1 ml dose (called A20 and A21). Additionally, both the PCV2 ORF2 and the SIV HA gene plasmid constructs were separately formulated with TGV200 PINC-polymer to yield 250 μg of each respective clone in a 1 ml dose (called T20 and T21). The final vaccine consisted of a four separate 1 ml 1M doses of each formulated clone. Table 2 shows all of the created vaccines, including the control vaccines.
The forty pigs were divided into four groups: Group 1 was administered 19×1 ml doses of A1-A19 Adju-Phos on Days 0, 21, and 42. Group 2 was administered 19×1 ml doses of T1-T19 TGV200 on Days 0, 21, and 42. Group 3 was administered 4×1 ml respective doses of A20, A21, T20, and T21 on Days 0, 21, and 42. Group 4, the negative control, had no treatment administered at all.
On day 56 of the study, pigs in groups 1, 2, and 3 were all administered the virulent SDSU#73 strain of PRRSV. The virulent SDSU#73 strain of PRRSV was diluted 1:10 in EMEM with 4% fetal bovine serum prior to administration to the pigs. A total of 2 ml of the diluted challenge virus was delivered intranasally to the appropriate pigs with 1 ml of the diluted virus administered to each nostril. The prechallenge and postchallenge titer of the PRRSV challenge virus was 104.59 TCID50/ml and 104.65 TCID50/ml, respectively.
The pigs were bled on days 0, 21, 42 and 56 of the trial to monitor seroconversion to vaccination. The pigs were also bled on days 57, 59, 61, 63, 66 and 70 of the trial to monitor seroconversion and viremia postchallenge. Clinical observations were recorded daily from day 54-70. On day 70, pigs were necropsied and gross lung lesions were recorded as percent lung involvement due to PRRSV. Pigs were weighed prior to the study start and on Days 56 and Days 70. A summary of the protocol in this example can be seen in Table 3.
In order to evaluate the results of the example, the main criteria used to determine the efficacy of the test treatments was the development of lung lesions characteristic of PRRSV. Serological response, postchallenge ADG, and rectal temperatures were evaluated as supporting criteria. The results of the example are summarized in Table 4.
There was no seroconversion to PRRSV as determined by the IDEXX PRRS ELISA in the any of the pigs receiving three doses of the respective A1-A19 Adju-Phos or T1-T19 TGV200 DNA vaccine prototypes. After virulent PRRSV challenge, there appeared to be a more rapid increase in the ELISA S/P ratios in the Group 2 than in the Group 1 pigs. Pigs in Group 3 (unvaccinated/challenged controls) had negative S/P ratios until exposed to virulent PRRSV challenge. The strict negative control pigs in Group 4 had negative S/P ratios throughout the study. The serology results are reported in Table 5.
The pig temperatures were monitored from two days prechallenge throughout fourteen days postchallenge. The baseline group average temperature throughout the challenge period for the Group 4 unvaccinated/unchallenged controls was 103.4° F.
The group average temperatures among all PRRSV-challenged groups were elevated at some point following challenge, however there was a temporal difference in days post challenge in which the group average temperature peaked. The group average peak temperatures in all groups challenged with PRRSV ranged from 105.9 to 106.1° F., whereas the negative controls group average peak temperature was 103.9. Group 3 showed a gradual increase in temperature postchallenge with a peak of 106.1° F. at nine days postchallenge. Groups 1 and 2 displayed a sharp increase in temperatures at two days postchallenge with peak temperatures of 105.9° F. and 105.6° F., respectively. Of note, the manner in which the postchallenge temperatures of the DNA vaccinated pigs in Group 1 and 2 rapidly increased, is similar to the manner in which PRRS KV vaccinated animals respond postchallenge. It has been observed that pigs vaccinated with experimental PRRS KV prototypes are usually “humorally-primed” (i.e. seropositive to PRRSV following vaccination). The animals vaccinated with these experimental PRRS KV prototypes also display a rapid increase in temperature soon after challenge, as was seen in this study with the DNA vaccinated pigs in Groups 1 and 2. This similarity in rapid temperature elevation following PRRSV challenge is an additional indication that the immune systems of the DNA vaccinated pigs were indeed primed to PRRSV antigen.
The Group 3 pigs displayed lung lesions characteristically found in a successful PRRSV challenge. The Group 4 pigs had no lung lesions at necropsy. The Group 1 and 2 pigs had group average lung lesions of 19.84 and 28.42, respectively. The lung scores for the individual pigs are reported in Table 6.
From these results, it is clear that DNA vaccines comprising various regions of the PRRSV genome can induce protection from virulent challenge in the respiratory model
This application claims the benefit of Provisional Application Ser. No. 60/581,350, filed on Jun. 17, 2004, the teachings and content of which are expressly incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 60581350 | Jun 2004 | US |
