Intranasal immunization with proteoliposomes protects against influenza

Intranasal immunization with proteoliposomes protects against influenza Nadia El Guink *:, Richard M. Kris *~,Gaff Goodman-Snitkoff*, Parker A. Small, Jr.* and Raphael J. Mannino *# Worldwide, influenza virus remains a serious disease wh&h has successfully eluded numerous attempts to design a consistently effective vaccine, ln part, these attempts have been thwarted because of a lack of basic understanding of the mechanisms which mediate protection and recovery from influenza infection. A better understanding of the roles of secretory antibody, serum antibody and cell mediated immunity vis-A-visprotection and recovery from influenza infection has allowed us more rationally to approach the design and administration of a vaccinefor influenza. We have constructed a vaccine composed of glycoproteins from the envelopes of either influenza of Sendai virus embedded in a lipid bilayer (immunosomes) mimicking the presentation of the virus to the cells during natural infection. Intranasal immunization with these immunosomes induces an adequate systemic Ir compared with intramuscular immunization and a superior local IgA response. These animals were specifically protectedfrom virus challenge. Keywords:Influenza; intranasal immunization; proteoliposomes

Introduction Influenza remains a serious and uncontrolled pandemic disease which kills more than 10000 people per year. Unfortunately, the ability of currently available influenza vaccines to protect against infection is exceedingly variable. In field trials the degree of protection following immunization has varied from 27 to 95% protection 1-4. Furthermore, annual appropriate immunization over 3 years in a school community demonstrated no overall protection. Following the first immunization ,-~50% protection was observed in exposed individuals. However, appropriate revaccination did not protect against subsequent infection and the overall attack was similar irrespective of immunization or lack thereofs. A better understanding of the host's system of defence to influenza may lead to an understanding of why the current vaccines are not always adequate and provide a more reasonable framework within which to design new vaccine. While serum antibody can prevent viral pneumonia and aid in the recovery from disease, it will not prevent upper respiratory infection in mice or ferrets 6-s. While cell mediated immunity is the key to recovery, local immunity, as manifested by secretory IgA, is responsible for protection from infection 1'8-~4. Thus, for protection from infection an appropriate vaccine for influenza should *Department of Microbiology and Immunology, Albany Medical College, 47 New Scotland Avenue, Albany, NY 12208, USA. tDepartment of Immunology and Medical Microbiology, Box J-266, JHMHC, College of Medicine, University of Florida, Gainsville, FL 32610, USA. :Present address: 6 Dr. Ibrahin AbdeI-Sayed Street, Greek Quarter, Bab-Sharkey, Alexandria, Egypt. SPresent address: Rorer, Inc., 680 Allendale Road, King of Prussia, PA, USA. #To whom all correspondence should be addressed. (Received 17 August 1988) 0264--410X/89/020147-05$03.00 @ 1989Butterworth & Co. (Publishers) Ltd

be designed to stimulate local antibody to prevent upper respiratory infection and serum antibody to prevent viral pneumonia. Therefore, we have attempted to mimic the structure and route of entry of the pathogen, through design of the vaccines themselves and the method of immunization. The vaccines were composed of liposomes containing purified influenza or Sendai (parainfluenza type 1) virus glycoproteins in the lipid bilayer and mimicked the appearance of a naturally occurring viral envelope. Additionally, we have compared the ability ofintranasal immunization, simulating the natural route of infection, to intramuscular immunization for their ability to produce local and systemic immunity and to protect against intranasal challenge with infectious influenza or Sendai virus.

Materials and methods Virus propagation Virus stocks were grown and purified essentially as described by Hsu et a115.Influenza (A/PRS/34) and Sendai (parainfluenza type 1) viruses were propagated in the allantoic sac of 10- or 11-day-old embryonated chicken eggs. Eggs were incubated with 1-100 egg infectious doses (103 to 105 viral particles as determined by HA titre) in 0.1 ml of phosphate-buffered saline. Eggs were incubated at 37°C for 48-72 h, followed by incubation of 4°C for 2448 h. Allantoic fluid was collected and clarified at 500g for 20 min at 5°C in a Damon (IEC/PR-J) centrifuge. The supernatant was then centifuged at 15 000 rev min- l for 60 min. This and all subsequent centrifugations were performed in a Sorvall PC2-B centrifuge at 5°C using a GG rotor. The pellets were resuspended in phosphatebuffered saline (PBS, pH 7.2) by vortexing and sonicating, followed by centrifugation at 2000g for 20 min. The Vaccine, Vol. 7, April 1989

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Intranasal proteoliposome immunization: Nadia El Guink et al.

two 2000g supernatants were combined and centrifuged at 15 000 rev min- 1 for 60 min. The resulting pellets were resuspended by vortexing and sonicating, aliquoted and stored at - 70°C. Sterile technique and materials were used throughout viral inoculation, isolation and purification.

10-fold dilutions) were inoculated into embryonated chick eggs. The eggs were incubated at 37°C and the allantoic fluid was harvested and tested for the presence of virus by HA of chick red cells.

Reconstitution of viral envelope liposomes

Results

Purified virus preparation (either influenza or Sendai virus) at a concentration of 2-5 mg of protein in 2 ml of PBS were mixed with 1 ml of 20% octyl-13-D-glucopyranoside (Sigma Chemical Co.) in PBS at 37*C for 20 min. The mixture was centifuged at 20 000g at 20"C for 1 h to remove the nucleic acid and nucleic acid associated proteins, while the lipid and membrane associated glycoproteins remained in the supernatant. Following centrifugation, the envelope and associated glycoproteins were reconstituted by dialysis against PBS to remove the detergent 16'17. The reconstituted liposomes are stable at 4"C and were used for immunization. Control liposomes were prepared by dissolving phosphatidylcholine:phosphatidylserine:phosphatidylethanolamine : sphingomyelin: cholesterol (1:1:2:1:5) (Avanti Polar Lipids, Inc.) in PBS with octyl-13-Dglycopyranoside and vesicles were prepared by dialysis against PBS to remove detergent.

Biochemistry of the virus and liposome preparations

Immunization and assay of protection Outbred mice were obtained from the New York State Department of Health and Laboratories and were immunized intranasally while awake, intranasally while anaesthetized (0.1 ml of 10 mg ml-' sodium pentobarbitol solution, i.p., for immunization of the total respiratory tract, TRT), or intramuscularly. The mice were immunized on days 1, 5 and 10 with 10 ~tg of viral protein in either 50 Ixl (for intranasal inoculation) or 100 lal (for i.m. inoculation). On day 15, either nasal wash and serum antibody levels were determined, or the mice were anaesthetized and challenged with 107 particles of live virus. Three days after the virus challenge, the mice were killed by cervical dislocation or with sodium pentabarbitol (0.15ml of 25 mg ml - i solution, i.p.). After sacrifice, the trachea and lungs of the mice were removed aseptically and placed in 2 ml of PBS, tritiated and stored at -70"C until titred. Antibody levels were determined by HI assay or by solid phase RIA with an assay slightly modified from that of Gerhard et aPs. A/Pr8/34 influenza virus vaccine, containing 64 HA units (0.05 ml) was immobilized on poly(vinyl chloride)microtitre plates (Scientific Products, Dynatech). The optimal dilution of the rabbit anti-mouse immunoglobulin reagents, to be used in the RIA, was determined to be a 1/32 000 dilution with the exception of the rabbit anti-mouse IgA which was determined to be a 1/16000 dilution. The standard used for serum antibody determination was a serum pool and the sensitivity of the assay for serum antibody was such that 0.1 units could be detected. The concentration of IgA antibody in nasal wash was much less than in serum. The standard was a pool of nasal wash derived from mice infected 23 days earlier. The sensitivity was such that 2 units was the lower level of detection. The IgG, IgM and IgA assays have not been standardized for comparable activity. However, the same assay was used to analyse IgA in serum and nasal wash.

Titration of virus from lungs To assay for the presence of infectious virus in the lungs, 0.1 ml of lung suspension (either undiluted, or 148

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A comparison of the biochemical properties of the viruses and liposomes derived from these viruses is presented in Table 1. Polyacrylamide gel electrophoresis in the presence of SDS and 2-ME of virus and viral liposomes revealed that the liposomes contained the respective envelope glycoproteins and had lost the nonglycosylated membrane protein and the proteins associated with the nucleocapsid complex (data not shown). The liposomes contain one third to one fifth the protein compared to the virus and about two thirds of the phosphate, indicating a retention of phospholipid, but loss of nucleic acid and phosphoproteins as demonstrated by polyacrylamide gel electrophoresis 19.Both purified viruses and their respective liposomes have haemagglutinating activity with chicken red blood cells, demonstrating that the liposomes contain some of the viral surface glycoproteins in a functionally active orientation. The five- to tenfold decrease in haemagglutinating activity observed when comparing reconstituted viral envelopes with intact viruses may be due to a number of factors. These include incomplete reconstitution of envelope proteins into the liposome bilayer or some protein denaturation during isolation and reconstitution. In addition, haemagglutination measures numbers of particles rather than the absolute reconstitution of active haemagglutinating molecules; a reduction in haemagglutinating activity can be a result of the reconstituted liposomes being larger (and therefore fewer) than the intact virions.

Infectivity of virus and liposomes Because the intent was to create a noninfectious vaccine, our preparations were tested for infectivity. The data (see Table 2) show that 106 influenza virus or Sendai virus particles were required to infect 50% of the mice, whereas 10 7 liposomes failed to infect the mouse lung. In a second experiment, various concentrations of infectious virus or liposomes were inoculated into embryonated eggs and the EIDs0 was determined. The data show that 102 virus Table I

Biochemical activities of reconstituted vesicles

Influenza virus (3.7 ml)

Reconstituted influenza vesicles (5.7 ml)

Sendai virus (1.8 ml)

Reconstituted Sendai vesicles (4.1 ml)

SDS electrophoretic analysis--proteins*

All viral proteins

Envelope (HA,NA)

All viral proteins

Envelope (HN,F)

Protein content (total rag) t

5.7

1.8

7.7

1.5

Phosphate content (total lag)*

116

73

126

89

Haemagglutination activity--total units ~

1.03 x 105

2.5 x 104

4.5 x 105

2.5 x 104

* By a modification of the Laemmli method =° ~Assayed by the Bartlett method 21 :Assayed by the Lowry method = §Determined by agglutination of chick red blood cells

Intranasal proteoliposome immunization." Nadia El Guink et al. Table 2

Biological activities of reconstituted vesicles Infectivity in mice (measured as IDso)

Infectivity in eggs (measured as EIDso)

mice immunized with influenzasomes are protected from infection with influenza virus but not from Sendai virus (p<0.001) and mice immunized with Sendaisomes are protected from infectious Sendai virus but not infectious influenza virus (p < 0.001). Thus demonstrating a specific, protective immune response.

Influenze virus

106 particles*

102 particles

Reconstituted influenza virus envelopes

Not detectable up to 10~ particles

Not detectable up to 4 x 10 '0 particles

Sendal virus

100 particles

2 x 102-4 x 102 particles

Protection o f nose and lung from infection

Reconstituted Sendal virus envelopes

Not detectable up to 107 particles

Not detectable up to 2 x 101° particles

In a separate experiment, mice were immunized intranasally while awake (nasal), intranasally while anaesthetized (TRT), or intramuscularly (i.m.), following the same schedule, with either inactivated influenza virus or with influenzasomes. They were either assayed for nasal and serum antibodies or were challenged with live influenza virus while anaesthetized. Table 6 shows the results of this experiment. Mice immunized with control liposomes had no detectable antibodies to the influenza glycoproteins in either serum or nasal wash. However, influenzasomes and whole virus vaccines stimulated an antibody response when immunization was either intramuscular or intra-

* Number of particles is calculated from the haemagglutination titre

Table 3 Serum antibody titres to influenza and Sendal virus* measured by haemagglutination inhibition Route of immunization Virus

Intranasal

Intramuscular

Influenza

1.45_+0.22

2.1 + 0

Sendal

1.5_+0.38

2.15_+0.13

*Titres are expressed as Ioglo Six mice per group were immunized with the homologous virosome (i.e. influenzasome or Sendaisome). Preimmune sera exhibited no haemaggiutination inhibition

particles were sufficient to infect 50% of the embryonated eggs, whereas 101° liposomes failed to infect the eggs. Therefore, the number of particles used to immunize mice in these experiments was shown not to be infectious.

Ability o f liposomes to stimulate an immune response To determine the immune response of groups of six mice to vaccination with influenzasomes or Sendaisomes, mice were immunized intramuscularly or intranasally while anaesthetized. The results of the experiments are presented in Table 3. Both intranasal and intramuscular vaccination of mice elicited serum HI responses against influenzae and Sendai viruses. The intramuscular vaccination elicited a higher and more consistent HI response against both viruses than did the intranasal vaccination.

Protection o f lungs from infection However, as shown in Table 4, this difference was not always reflected in better protection against influenza virus or Sendai virus challenges. No virus is recovered from the lungs of unchallenged mice, whereas six of seven nonimmunized control mice had virus in their lungs 3 days after challenge (log10 EIDs0, 3.8 _+0.75 for influenza and 3.67 _+0.82 for Sendai for six animals with infection). The mice immunized with influenzasomes either intranasally or intramuscularly were all protected from virus challenge and no infectious virus was recovered (p<0.01). Intranasal immunization with Sendaisomes protected six of seven mice from challenge with infectious virus (p < 0.05), while intramuscular vaccination with Sendiasomes protected only five of seven mice from challenge (p < 0.05). Therefore, we can conclude that both intramuscular and intranasal immunization afford pulmonary protection to mice against intranasal challenge with the appropriate infectious agent. This immunity is specific as can be seen in Table 5. Anaesthetized mice were immunized intranasally with either influenzasomes or Sendaisomes and then challenged with infectious influenza or Sendai virus. As can be seen,

Table 4

Protection of mice by immunization with virosomes* Route of immunization

Virus

None

Intranasal

Intramuscular

Influenza

3.8_+0.75(6/7)

N.V?(0/7)(p<0.01)

N.V.(0/7)(p<0.01)

Sendal

3.67±0.82(6/7)

1_+ 0(1/7) (p<0.05)

2_+ 0(2/7) (p< 0.05)

*Mice were immunized with the homologous virosomes and infectious virus in the lung extracts was titred 3 days after awake intranasal challenge, The results are reported as Iogl0EIDsofor lung extracts of mice containing virus. No virus was recovered from unchallenged mice tNumber of mice with infectious virus/number of mice challenged ~No virus recovered from lung extracts of these mice

Table 5 Specificity of protection produced by intranasal immunization with virosomes Challenge virus Immunization

Influenza

Sendal

None

1.86-+ 0.90(7/7)

1.57-+ 0.78(7/7)

Influenzasome

No virus (0/7) (p< 0.001)

1.86-+0.90(7/7)

Sendaisome

2.57 -+ 1.72(7•7)

No virus (0/7) (p< 0.001)

Anaesthetized mice were immunized intranasally with one virusome preparation and then challenged intranasally with either influenza or Sendal virus, Infectivity of the lung extracts is expressed as Iogl0EIDs0

Table 8

Antibody and protection induced by liposomes Serum

Protection ~ No immune/ total

Vaccines

Nasal wash* igA

Flusomes TRT Nasal I.M.

40(___49) 56(___43) 100(__.61) 58(___25) 7/7(p<0,001) 42(__.16) 29(___46) 38(__.39) 15(_+16) 7/7 (p< 0,001) 4(_+5) 61 (_+30) 56(_+39) 30(-+23) 3/7(p<0.05)

IgG

IgM

IgA

Whole virus* TRT 128(+123) 54(-+39) 129(4-77) 80(__.24) 7/7(p<0.001) Nasal 70(_+63) 31(-+28) 158(_+105) 165(_+170)7/7(p<0.001) I.M. 21(_+18) 100(4-132) 125(_+71) 119(4-91) 7/7(p<0.001) Control Liposomes - -

0/7

*All antibody concentrations were determined by radioimmune assay tChallenge was of total respiratory tract tA/Pr8/34 vaccine was a gift from Lilly Labs (Indianapolis, IN, USA)

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nasal. Both vaccine preparations produced a significantly greater local immune response (i.e. detectable in the nasal wash) when given intranasally, the common route of infection, than did intramuscular immunization. Animals immunized with control liposomes were not protected from intranasal challenge with live influenza virus and whole inactivated virus protected the mice from virus challenge independent of the route of immunization. When mice were immunized with influenzasomes, intranasal immunization was superior to intramuscular in that all the mice were protected from viral challenge (nasal-7/7, TRT-7/7, i.m.-3/7). These results support the hypothesis that local secretory antibody is a significant factor protective for upper respiratory infections such as influenza.

Discussion The results presented here demonstrate that intranasal immunization with virosomes provides a high degree of protective immunity. While both intranasal and intramuscular immunization elicited antibody production to homologous virus as measured by haemagglutination inhibition, the serum antibody titres were always greater following intramuscular immunization. However, the difference in serum antibody titre did not correlate with protection. The results reported here demonstrate that significant levels of secretory IgA are produced and may be recovered from nasal secretions following intranasal and total respiratory tract immunization with influenzasomes. In all cases, protection of mice immunized via the intranasal route was as good as, or better than, that following intramuscular immunization and correlated with increased IgA in the nasal wash. As compared to intramuscular immunization, intranasal immunization selectively enhances local secretory immunity without compromising serum antibody production and intranasal immunization with influenzasomes is able to offer significantly more protection from infection by influenza virus than is afforded by intramuscular immunization. There are two explanations for the success of our preparations. The first is the method of vesicle preparation. Previously, it has been observed that immunization with subunit vaccines composed of isolated glycoprotein spikes has been disappointing. Single peplomers of glycoproteins are poorly immunogenic and render little or no protection against viral challenge, while micelles of the spike proteins vary significantly in their ability to evoke a protective immune response, and often require the presence of adjuvants to achieve significant levels of protection. However, it has been shown that when viral glycoproteins are integrated into a lipid bilayer they are more immunogenic than in the absence of lipid (for review see Ref. 23, also Refs. 2, 24-29). This is most likely due to the insertion of the viral glycoproteins into the lipid membrane, thus orienting them in a natural configuration vis-h-vis the immune system 2'24'26-35. We have produced liposomes that contain viral glycoproteins in a biologically active configuration, demonstrated by the high levels of haemagglutination activity in these preparations, and lacking the core proteins and nucleic acids normally associated with heat or formalin inactivated vaccines. Data presented here show that liposomal vaccine particles are not infectious for mice or embryonated chick eggs to our testing limits, demonstrating the intrinsic safety of this type of vaccine preparation. Therefore, the results presented here demonstrate that incorporation of viral 150

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glycoproteins into liposomal membranes induces protective levels of secretory and serum antibody without the risk of infection or side effects normally associated with inactivated vaccines. Second, the novel, intranasal, route of immunization results in significant antibody secretion at the natural site of infection. This factor appears to be important in the protection of populations at risk for influenza, and perhaps for other diseases such as respiratory syncitial virus, cholera and herpes simplex virus where high circulating antibody titres do not always correlate with protection. For these diseases, it is the presence of local secretory antibody which is protective 3~2. As shown here, intranasal administration ofliposomal vaccines results in local secretory antibody levels only slightly lower than those obtained with intranasal administration of whole inactivated virus. However, the intranasal levels of antibody obtained are 10 times those obtained with intramuscular immunization with liposomal vaccines and twice those obtained with intramuscular immunization with whole inactivated virus. Furthermore, the levels of secretory antibody from liposomes are sufficient to afford protection to all the mice challenged with infectious virus, suggesting that local specific protective immunity is produced as a result of intranasal immunization with liposomal vaccines. In fact, intranasal administration of the vaccine provides greater protection than does the intramuscular administration, a finding which confirms previous work with inactivated and attenuated viruses in a number of species 39,43,44.45. TO conclude, data presented here indicate that intranasal vaccination with liposomes containing only the surface glycoproteins of the influenza or Sendai virus produces both a local secretory and a circulating immune response and protects against the homologous but not heterologous virus, showing the specificity of the vaccine preparations. Thus, liposomes containing surface glycoproteins of influenza or Sendai virus administered intranasally are effective in eliciting an immune response and protecting mice against infection by the homologous virus. These findings support the concept that liposome based subunit vaccines can be used to develop protective immunity against infectious agents whose primary route of infection is via mucosal surfaces and for which no effective vaccines are currently available.

Acknowledgements The authors would like to thank Maureen Cavanaugh and Carole D'Aloia for their assistance in preparing the manuscript.

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