Immune Response to Parenteral and Oral Measles Vaccine Combined with Adjuvants

H. Mirchamsy DVM, A Shafyi DVM, H. Mansouri MSc, P. Ahourai DVM,

Z. Hamzelou BSc, N. Naseri BSc, G. Fateh BSc

The Razi Inistitute, Tehran, Iran

Abstract Background—Approaches to mucosal immunization against measles have been mostly centered on intranasal or inhalation routes. Methods—Immune responses of vervet monkeys immunized parenterally or orally with human doses of live attenuated measles vaccine alone, or in combination with an adjuvant are compared. Polar glycopeptidolipids of Mycobacterium chelonae and negatively charged liposomes were used as adjuvants. Measles antibody was determined by hemagglutination-inhibition test. Results—Comparison of HI titers revealed that PGPL-MC as an adjuvant, significantly potentiated the HI antibody response in monkeys parenterally immunized with live attenuated measles vaccine. On the contrary, liposomes as adjuvant did not improve the HI titer. Monkeys immunized orally with 2 ml of liposomes and boosted one month later, responded by low titers of 50%, and 37%, respectively. It was found that neutralization of the gastrointestinal tract enzymes by the antienzyme aprotinin before oral immunization was ineffective. Conclusion—It is concluded that live attenuated measles vaccine adjuvanted with PGPL-MC and administered parenterally has a high immunostimulatory capacity whereas oral route is probably ineffective for mass immunization against measles infection.

Introduction

Oral immunization against measles infection has been studied by several investigators. Black and colleagues failed to vaccinate 10 children by applying cotton swab to the oral mucosa. In a limited study, Cernescu et al, using L16 attenuated strain of measles virus adapted to human diploid cells (HDC), obtained 47% seroconversion by feeding the vaccine with sucrose, and 76% by oral spray. In another limited trial, Whittle et al, using 2 doses of Edmonton-Zagreb (EZ) vaccine through a large particle spray, within a 6-week intervalcould achieve only 10% immunization in the children under the study. In a review of alternative routes for administration of measles virus, Cutts et al, concluded that the oral route was probably ineffective and large field trials had yet to be conducted. However, to date there is no published study of an orally administered vaccine in an enteric-coated capsule that might be protected during the passage down the gastrointestinal tract. Works in this area are, however, currently underway in Russia.¹¹ The first experimental study on the efficacy of enteric coated capsules of measles vaccine has been reported by Nechaeva et al⁶ who claim that tablets of live virus vaccine by oral administration would cause stimulation of humoral and cellular immunity in a majority of laboratory animals.

Materials and Methods

Measles vaccine

Live attenuated measles vaccine of the Aik strain, grown in C.F. Cells lot 1125-1, produced locally, was applied. Ten-dose vials of lyophilized vaccine were used containing 5.25 log 10 of virus per vial.

Methods

Monkeys were given aprotinin, orally, followed by an oral dose of live attenuated measles vaccine, using either polar glycopeptidolipids of Mycobacterium chelonae (PGPL-MC) or negatively charged liposomes as immuno-stimulants. Serum measles antibody was determined using hemagglutination inhibition (HI) test.

Products

PGPL-MC: This adjuvant was kindly provided by Professeur Pilet, from the Ecole Nationale Vétérinaire d'Alfort-in-France. Each vial contained 35 mg of the product. The content of each vial was dissolved in double distilled water as indicated in the experimental design.

Liposomes: The negatively charged liposomes were produced with egg lecithin and cholesterol following the method described by Deamer and Bangham.⁹ The product contained a mixture of unilamellar and multilamellar vesicules.

Inhibition of gastro-intestinal (GI) enzymes

Monkeys received, 1 ml or 10000 kIU of aprotinin, a natural low molecular mass polypeptide protease inhibitor, before oral vaccination.

Animals

African green monkeys or vervet (Cercopithecus aetiops) weighing 1.5-2 kg were imported from Tanzania. The monkeys used in this study lacked detectable measles HI antibody.

Collection of Serum

Blood from monkeys was collected by venous puncture. Sera were stored frozen at -20° c until titrated.

Serology

Measles antibody was determined by hemagglutination-inhibition (HI) test.

The test was carried out according to the method recommended by WHO.⁷ We used 4 hemagglutinin (HA) units in all tests of this study, in order to determine the inhibitory activity of the adsorbed sera. In case of negative response, the tests were repeated by reducing HA units to two or even one unit to detect low serological responses.

Experimental Designs

Vials of lyophilized vaccine were diluted in sterile and chilled double distilled water (DDW), and kept at +2° c until use. For oral administration, vaccines were diluted in 3-4 ml of a 10% solution of sucrose in DDW.

The monkeys were under veterinary surveillance for local and general reactions. Four weeks after immunization, a booster dose, similar to that in the primary immunization, was administered to all monkeys. Sampling was accomplished one month after introducing the booster dose.

Immunization Protocols

Immunization with measles vaccine mixed with PGPL-MC: In the first study, three groups were selected each comprised of 8 monkeys. All monkeys in the first group were immunized parenterally and boosted with one human dose of measles vaccine without adjuvant. The monkeys in the second group were immunized parenterally and boosted with one human dose of measles vaccine mixed with 3.5 mg of PGPL-MC. The third group was immunized and boosted orally with 10 human doses of measles vaccine mixed with 35 mg of PGPL- MC.

Immunization with measles vaccine mixed with liposomes: In the second study, two groups were selected, each comprising 8 monkeys. Monkeys of the first group were immunized and boosted parenterally with one human dose of measles vaccine mixed with 1 ml of liposomes, and those of the second group were immunized orally with 10 human doses of measles vaccine mixed with 2 ml of liposomes.

Oral immunization of monkeys after inhibition of GI enzymes: In the third study, two groups each composed of five monkeys were treated as follows:

Results

Table 1 shows the results obtained from different treatments. The mean titers were significantly different (Kruskal-Wallis, p < 0.0001). It was noted that PGPL-MC significantly potentiated the HI titers recorded in the first study. On the contrary, oral immunization and boosting with 10 human doses of measles vaccine adjuvanted with 10 fold higher PGPL-MC, initiated a low HI titer in 50% of monkeys. It was also found that monkeys immunized parenterally with measles vaccine adjuvanted with PGPL-MC, showed a high HI response 4 months later as compared with those immunized without adjuvant.

Comparing the results of the first and the second studies, it was noted that liposomes did not improve the HI antibody titer. The oral use of liposome as an adjuvant was not effective, since the serological responses were either low and probably not protective, or else disappeared a few weeks later.

The serological responses, in the last study, were very low (detected by using 2 HA antigen units) and these low responses were found to disappear within a few weeks.

Discussion

In the present study, it was found that parenteral administration of a single human dose of live attenuated AIK measles vaccine, adjuvanted with 3.5 mg of GPLP-MC and followed by a booster dose exhibited high HI antibody titer of up to 8 folds one month later, in monkeys, when compared to the control vaccine. This finding is similar to the observation of Gjata et al,⁴ who have reported high stimulation of HI titer in mice following injection of inactivated influenza vaccine combined with a small amount of GPLP-MC. Due to the digestion by GI enzymes, oral vaccination requires a high dose of the immunogen. Furthermore, on account of the large size of the mucosal surface of the GI tract, the amount of the adjuvant used, had to be adequate in order to reach the entire mucosal surface, as reported in a previous study.⁹ In this study, although 10 doses of live measles vaccine mixed with 10 times more GPLP-MC were administered orally, the immune response was merely a weak stimulation of circulating HI antibody when compared with those used in parenteral assay.

Liposomes which have been experimentally shown to be convenient immuno-adjuvants for stimulation of mucosal and systemic antibody response against inactivated measles virus by intranasal administeration in mice,⁵ disclosed disappointing results in our parenteral and oral assays in monkeys.

Finally, the oral use of live measles vaccine alone after neutralization of GI enzymes by anti-enzyme which was found to be effective in one of our previous reports,^8,10 proved to be inefficient in the case of live measles virus vaccine.

We believe that this study provides evidence for high immuno-stimulatory capacity of polar glycopeptidolipids of M. chelonae mixed with live attenuated measles vaccine, when administered parenterally. In oral immunization, live measles vaccine mixed with GPLP-MC or with liposomes appears to be degraded in the GI tract and there is thus little tropism for mucosal inductive sites. Consequently, oral route is probably ineffective for mass immunization against measles infection.

Aknowledgments

The authors are grateful to Professeur Charles Pilet, Directeur, Institut d'immunologie Vétérinaire et Comparée, Ecole Vétérinaire d'Alfort-France for providing the polar glycopeptidolipids of mycobacterium chelonae. We wish

also to thank Mr. Elikay for his kind cooperation.

References

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