Virus Research, 16 (1990) 83-94 Elsevier
VIRUS
83
00570
Comparison of inactivated, live and recombinant DNA vaccines against influenza virus in a mouse model Paul A. Rota, Barun K. De, Michael W. Shaw *, Renee A. Black, William C. Gamble and Alan P. Kendal Influenza Branch, Division of Viral and Rickeitsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, Atlanta, GA 30333, U.S.A. (Accepted
13 December
1989)
Summary
The protective efficacy of influenza hemagglutinin expressed from recombinant vaccinia virus was compared with that induced by inactivated or infectious influenza vaccines. Intraperitoneal and intranasal routes of vaccination were compared. All the vaccines except the intranasally administered, inactivated vaccine induced detectable levels of neutralizing and hemagglutination-inhibiting antibodies in the serum of mice at 28 days postvaccination. Immunization with any of the intranasally administered vaccines reduced the amount of influenza virus nucleoprotein antigen in lungs after challenge with a homologous, mouse-adapted strain of influenza virus. Intraperitoneally administered vaccines failed to provide such protection. These results indicated that the route of vaccine administration may be the most critical factor for inducing protective immunity. The results also showed that in this mouse model a recombinant DNA-based vaccine could provide protection equivalent to that provided by conventional attenuated and inactivated influenza vaccines. Influenza virus; DNA vaccine; Mouse model
Correspondence ro: P.A. Rota, Influenza Branch, G-16, Centers for Disease Control, Atlanta, GA 30333, U.S.A. * Present address: M.W. Shaw, Department of Epidemiology. University of Michigan School of Public Health, AM Arbor, MI 48109, U.S.A. 0168-1702/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
84
Introduction Several approaches have been evaluated in an attempt to develop improved vaccines against influenza viruses, an effort complicated by the continual antigenic variation of the virus. Currently only inactivated virus vaccines composed of purified whole virus, chemically disrupted virions or purified subunit components have been licensed for use in the United States. Live attenuated vaccines have been widely used in a few countries and are undergoing clinical evaluation in the United States (Kendal et al., 1981; Couch et al., 1985). In theory, subunit vaccines could be prepared by using recombinant DNA expression systems. For example, human influenza virus hemagglutinin (HA) has been expressed from recombinant vaccinia virus and been shown capable of eliciting hemagglutination-inhibiting (HI) antibody in rodents. These animals were partially (Smith et al., 1983) or fully protected (Small et al., 1985) after challenge with homologous strains of influenza virus. Noninfectious vaccines containing the HA (Kuroda et al., 1986; De et al., 1988) or infectious vaccines containing the neuraminidase (Webster et al., 1988) of avian influenza viruses that were derived from baculovirus or vaccinia virus expression vectors have protected chickens against lethal challenge with virulent avian influenza viruses. However, no studies have yet been reported that directly compared recombinant DNA-derived influenza vaccines with traditional inactivated or live attenuated vaccines. Additionally, the route of administration has been suggested to be an important determinant in the efficacy of influenza vaccines (e.g. Small et al., 1985). While vaccination by the parenteral route is best for the induction of a humoral response, vaccination in the upper respiratory tract may be preferred for inducing a protective local antibody response against a respiratory virus (Liew et al., 1984). Therefore, we have compared the relative protection seen in a mouse model system after vaccination by the intranasal or parenteral routes with inactivated, live, and recombinant DNA-derived vaccines containing the same influenza type A hemagglutinin. To evaluate vaccine efficacy, we have measured both serum neutralizing antibody and virus production in the affected organs after challenge with a mouse-adapted strain of influenza virus.
Materials and Methods Viruses and vaccines Influenza viruses were cultivated in embryonated hen’s eggs. Influenza A/Ann Arbor/6/60 (H2N2) wild-type (WT) virus was from the stocks of the World Health Organization Influenza Center (WHOIC) at the Centers for Disease Control (CDC) in Atlanta, Georgia (originally obtained from Dr H.F. Maassab, University of Michigan). Two mutants of this virus, cold-adapted (CA) A/Ann Arbor/6/60 and mouse-adapted pathogenic (MA) A/Ann Arbor/6/60 were also kindly provided by Dr Maassab. The MA virus had been passaged in mouse lungs 44 times before being
85
amplified in the allantoic cavity of emb~onated hens’ eggs prior to use in subsequent experiments. The infectivity of the final egg-derived MA virus was assayed in mice before the challenge experiments. The purification of viruses and virion RNA were as previously described (Palese and Schulman, 1976; Cox and Kendal, 1984). Sequencing Sequences of the influenza HA genes were obtained from viral RNA by using a modification of the Sanger dideoxy method (Chen and Seeberg, 1985; Robertson et al., 1985). Primers used in the sequencing reactions have been described by Cox et al. (1988).
Vaccines We constructed recombinant vaccinia viruses expressing the HA of A/Ann Arbor/6/60 by using previously described techniques (Mackett and Smith, 1986; Rota et al., 1987; De et al., 1988). The expression of authentic influenza HA was verified by Western blot analysis of lysates prepared from cells infected with the recombinant virus (Tsang et al., 1983) by using rabbit hyperimmune antiserum against whole A/Ann Arbor/6/60 virions. Cell surface localization of the HA expressed from the recombinant vaccinia virus was also verified by immunoelectron microscopy (De, unpublished observations). Recombinant vaccinia virus vaccine was purified and titrated by plaque assay as previously described (De et al., 1988). For preparation of inactivated influenza vaccine, ~adient-puffin A/Arm Arbor,/6/60 WT virus was resuspended in phosphate-buffered saline (PBS) containing 0.15% formalin. After a 48 h incubation at 4”C, the virus was pelleted from the formalin-containing buffer (100,000 x g, 1 h, 4°C) and resuspended in PBS. Inactivation was monitored by titrating infectivity in MDCK cell cultures. No plaque-forming units were detected in standard plaque assays of the inactivated vaccine preparation at any dilution up to concentrations causing cytopathic effect solely from the input inoculum. Commercially produced inactivated influenza vaccines were used as negative controls in these infectivity assays. Protein concentration was determined by using the Lowry assay (Lowry et al., 1951).
Animal ~~ccinati~~ and ~~a~len~eexperiments Vaccines were administered to female A/J mice 5-7 weeks old by either the intranasal or the intraperitoneal route. Intrauasal vaccines were given in a lo-p1 volume under CO, anesthesia. Intraperitoneal vaccines were given in a volume of 0.5 ml. Serum samples for antibody titration were obtained by sacrificing groups of five mice and exsanguinating them at intervals after vaccination. The concentration of neutralizing antibody pooled serum samples was determined by using the microneutralization assay described by Harmon et al. (1988). Mice were challenged with the MA A/Arm Arbor/6/60 virus (FlM44E3). Since the level of anesthesia has been shown to effect the outcome of influenza infection
86
(Yetter et al., 1980), mice were individually anesthetized with CO, and infected intranasally with approximately 1 X lo4 mouse ID,, of the MA virus in a volume of 10 ~1. Lung homogenates were prepared from groups of five mice at 2, 3,4, 5,8, and 10 days postchallenge as described previously (Rota et al., 1987). The concentration of influenza type A nucleoprotein (NP) antigen present in the pooled lung homogenates was determined by using time-resolved fluoroimmunoassay (TR-FIA) (Walls et al., 1986). All samples were run in triplicate and the results were corrected for lung weights.
Results
HA sequence changes associated with mouse adaptation
Because it is known that laboratory passage can select for different subpopulations of influenza virus, the sequences of the A/Ann Arbor/6/60 HAS in the vaccine and challenge viruses were compared by direct sequencing of viral RNA. A total of 11 changes was noted when the deduced amino acid sequences of the HA genes of the WT, CA, and MA A/Ann Arbor/6/60 viruses were compared (Table 1). Of these, nine were in the HA1 polypeptide. None occurred in the known major antigenic sites. Changes at five positions (53, 71, 180, 221, and 299) would cause significant changes in local hydropathy. Also, the changes at positions 180 and 221 occur in an area close to the receptor-binding pocket of the HA. However, as presented in the following, these differences did not seem to interfere with the generation of protective immune responses in test animals.
TABLE 1 Changes in the amino acid sequences of the hemagglutinins of the cold-adapted and mouse-adapted influenza A/Ann Arbor/6/60 viruses Position
Wild type
CA’
MA2
18 53 71 151 180 199 221 264 299 367 378
Asn
Be
Asp Ser
Asp Ser
Ile Val
LYS Ile
LYS Asn
Gly A% LYS Glu
G’Y
Lys Ala
’ Cold-adapted. 2 Mouse-adapted (pathogenic).
GlY LYS Gly LYS Ser
Arg Asn Ile Val A% Gln Glu A% Ser
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Response to vaccination To determine the optimum dose for each vaccine, we inoculated mice with serial dilutions of each of the experimental influenza vaccines. Mice to be used in challenge studies were given a dose of vaccine equivalent to 50 times the highest dilution that had induced detectable serum neutralizing antibodies. However, for the inactivated, intranasal vaccine, the dose given was the same as that used for intraperitoneal vaccination. The doses used and the levels of neutralizing antibodies induced at 30 days postvaccination are shown in Table 2. All the vaccines induced detectable levels of neutralizing antibodies in serum except for the intranasally administered, formalin-inactivated influenza virus.
Protection studies A/J mice were selected for our study because, of four inbred strains tested, they were the most susceptible to infection with the particular mouse-adapted influenza A virus we were using (results not shown). Groups of mice immunized as described earlier were challenged intranasally with MA A/Ann Arbor/6/60 virus 30 days after vaccination. Protection was evaluated by measuring with TR-FIA the reduction in yield of influenza A virus NP antigen in the lungs at various times after challenge (Fig. 1). Results from this method have been found to correlate well with results from plaque assays on MDCK cell monolayers (Rota et al., 1987, and unpublished results). The controls for each challenge experiment consisted of mice vaccinated with PBS or with WT vaccinia virus (New York Board of Health strain). Initially, vaccination with live CA A/Ann Arbor/6/60 virus vaccine or live WT (not mouse-adapted) virus given intranasally was compared to intraperitoneal vaccination with recombinant vaccinia virus expressing the H2 HA or inactivated influenza virus vaccines (Fig. 1).
TABLE
2
Experimental
influenza
type A (H2N2)
vaccines
Vaccine
Dose/mouse
Route
Live vaccines A/Ann Arbor/6/60 (WT) A/Ann Arbor/6/60 (CA) vaccinia A/AA-HZHA vaccinia A/AA-HZHA
2.5 5.0 4.0 1.0
IN IN IP IN
800 200 200 100
Formalin-inactivated A/Arm Arbor/6/60 A/Ann Arbor/6/60
1.3 pg HA 1.2 pg HA
IP IN
100 < 25
’ Serum neutralizing
vaccines (WT) (WT)
x x x x
lo6 10’ lo5 lo6
EID,, EIDs, PFU PFU
Titer ’
antibody titer vs. A/Ann Arbor/6/60 (wt) at 30 days post-vaccination. average of three replicates of each serum pool (Abbreviations: PFU, plaque-forming intranasal; IP, intraperitoneal; EID, egg infectious dose).
Number is units; IN,
88
6.5 6.25
5.75
6
5.5
5.75 5.5
5.25
5.25
5
5
4.75
4.75 4.5
4.5
4.25
4.25
4
4
3.75 3.5
3.75
3.25
3.5
3
3.25
2.75 , 0
, 1
, 2
, 3
, 4
, 5
, 6
, 7
, 8
, 9
1
10
’
2.5 d
1
i.
3
4
5
6
7
8
9
10
Fig. 1. Comparison of vaccination with recombinant vaccinia virus or inactivated influenza virus by the intraperitoneal route and vaccination with live influenza virus by the intranasal route. Mice were vaccinated with formalin-inactivated (filled triangle), CA (asterisk), WT (open square) A/Ann Arbor/6/60 virus or recombinant vaccinia virus expressing the H2 HA (X), or PBS (filled square). PBS, formalin-inactivated and recombinant vaccinia virus were given intraperitoneally; WT and CA viruses were given intranasally. Figure shows the amount of influenza NP antigen (as log,, of TR-FIA counts per minute (cpm)) in lung homogenates (y axis) at 0 through 10 days postchallenge with MA virus (x axis). Mice were challenged 30 days (Panel A) or 3 months (Panel B) after initial vaccination.
The levels of influenza NP antigen detected in the control groups after challenge were nearly identical at all the time points tested. Because vaccinia virus alone did not induce non-specific antiviral activity in the mice only the antigen levels for the PBS-vaccinated control mice are shown. In these control mice, NP antigen was detected at 3 days postinfection and peaked at 4-6 days postinfection; antigen levels returned to background levels by 8 days postinfection. Mice receiving any of the vaccines except the intranasally administered inactivated virus had neutralizing antibodies in their serum at the time of challenge (Table 2). However, only the intranasally administered, live influenza viruses significantly reduced the amount of influenza virus NP antigen detected in the lung homogenates after challenge (Fig. lA,B). Protection from these intranasal vaccines was nearly complete; antigen levels rose only slightly above background readings at day 0, and all groups had at least a 97% reduction in detectable antigen compared with the peak levels detected in control animals. Similar levels of antigen reduction were observed when vaccinated mice were challenged at 3 months postvaccination (Fig. 1B). In contrast, the single dose of formalin-inactivated vaccine given intraperitoneally failed to reduce virus antigen levels in lung homogenates, and peak antigen levels in these animals were as high as or higher than in the control animals whether challenge was 1 or 3 months after vaccination (Fig. lA,B). In addition, an increase in antigen was consistently detected earlier in these animals than in controls. An intermediate level of protection was produced by intraperitoneal inoculation of the recombinant vaccinia virus expressing the H2 HA. Although a rise in NP
89
..._ \
i
4.5 / I
4.25
\
3.25
3’
, 0
, 1
, 2
, 3
, 4
, 5
, 6
, 7
, 8
(
9
,
’
IO
Fig. 2. Results of vaccination by the intranasal route. Figure shows the amount of influenza nucleoprotein [as log,, of TR-FIA counts per minute (cpm)] in lung homogenates at 0 through 10 days after challenge with MA virus. Mice were challenged at 30 days postvaccination. Symbol assignments are the same as those for Fig. 1 except that the formalin-inactivated and recombinant vaccinia virus vaccines were given intranasally.
antigen was detected, peak antigen levels in mice vaccinated with this recombinant vaccinia virus were lower than peak antigen levels in control animals whether challenged at 1 or at 3 months postvaccination (Fig. lA,B). To test the effect of the route of administration on the ability of the vaccines to protect against influenza challenge, mice were vaccinated with the formalin-inactivated and the recombinant vaccinia virus vaccines by the intranasal route before challenge (Fig. 2). Only mice given the recombinant vaccinia virus developed detectable serum neutralizing antibodies when vaccinated by this route (Table 2). However, when either group of mice was challenged with the MA A/Ann Arbor virus, both were highly protected. This protection, though not complete, was nearly equivalent to the protection provided by the intranasally administered live WT A/Ann Arbor influenza virus. All the vaccinated mice had at least a 97% reduction in the amount of peak antigen levels compared to control mice.
Discussion The primary purpose of this investigation was to expand on earlier studies that had examined the efficacy of vaccination with recombinant vaccinia viruses expressing HA. It had been shown that recombinant vaccinia viruses expressing the H3 HA provided partial protection when given parenterally (Smith et al., 1983) and full protection when administered intranasally (Small et al., 1985). In contrast, parenterally administered recombinant vaccinia virus expressing the influenza B HA gave full protection against challenge with a non-mouse-adapted strain of influenza B (Rota et al., 1987,1989). A study by Stott and coworkers (1987) showed that mice vaccinated with vaccinia recombinants expressing RSV G protein developed equiv-
90
alent levels of serum antibody after intranasal or intraperitoneal vaccination. However, the levels of antibody in the lungs of the intranasally vaccinated mice were more than lo-fold greater than in mice vaccinated by the intraperitoneal route. The results of the challenge experiments presented here confirmed that the route of administration was more important for the induction of protective immunity than the actual composition of the vaccine. Presumably, only low levels of local antibody were induced in the respiratory tract by intraperitoneal vaccination, and these levels were insufficient to provide complete protection against the mouse-adapted influenza virus used for the challenge in spite of the presence of neutralizing antibody in the serum. The role of local immunity in the upper respiratory tract is emphasized by the response of mice vaccinated with intranasally administered, inactivated vaccine: serum antibody was essentially undetectable prior to challenge, yet the animals were protected. Results of challenge experiments may differ according to the degree of adaptation or pathogenicity of the challenge virus. The measures of virus antigen depicted in Figs. 1 and 2 include only the lower respiratory tract; the entire lung, trachea and bronchi were excised and assayed. It may be that the apparent unrestricted growth of the virus after challenge of intraperitoneally immunized mice was limited to the trachea, and the mice were protected against a potentially fatal pneumonia. It has been clearly shown in the mouse system that serum antibody prevents viral pneumonia but not tracheitis or rhinitis (Loosli et al., 1953; Ramphal et al., 1979; Small et al., 1985). In our study, all the vaccinated animals survived, whereas lo-30% of the control animals died, depending on the experiment. These levels of viral pathogenicity and vaccine protection may be more relevant to human influenza than are the levels of pathogenicity and protection observed in model systems where highly lethal influenza viruses are used for challenge. At this time, we have no explanation for the finding that the peak levels of influenza antigen detected in the lungs after challenge were greater in mice that had been vaccinated by the intraperitoneal route than in the unimmunized control animals. Antibody enhancement of viral infectivity may be considered. Enhanced uptake by macrophage-like cells of antibody-coated influenza virus has been implicated in some other systems, possibly through Fc receptor-mediated phagocytosis (Webster and Askonas, 1980; Ochiai et al., 1988). This phenomenon has also been observed after infection with other viruses such as Venezuelan equine encephalitis virus (Levitt et al., 1979), yellow fever virus (Schlesinger and Brandriss, 1981), and reovirus (Burstin et al., 1983). Most of the influenza studies have shown a direct inverse correlation between influenza vaccine-induced serum antibody levels and disease incidence (reviewed in Salk and Salk, 1977). The roles of local humoral and cellular immunity are not fully understood and may differ when vaccines are administered intranasally or parenterally. It has been reported that nonreplicating virus administered intranasally was capable of inducing local immunity (Kasel et al., 1968). Later studies on the effects of intranasal, inactivated influenza vaccines showed greater protection from aerosolized vaccine than from the same vaccine given subcutaneously (Waldman et al., 1969; Waldman and Coggins, 1972; Zahradnik et al., 1983). However, other studies
91
on humans have failed to confirm the advantage of local over parenteral immunization with inactivated vaccine (Gwaltney et al., 1971; Edmonson et al., 1971; Wenzel et al., 1973). Despite the many potential drawbacks pertaining to the use of artificial models for human influenza, the observations reported here suggest a need for further studies of vaccination by the intranasal route. These studies should include inactivated vaccines and live attenuated virus as well as antigens produced by recombinant expression systems.
Acknowledgement
The authors are grateful to Jennifer Rota for assistance with the TR-FIA assays.
References Burstin, S.J., Brandriss, M.W. and Schlesinger, J.J. (1983) Infection of a macrophage-like cell line, P288D1, with reovirus: effects of immune ascitic fluids and monoclonal antibodies on neutralization and on enhancement of viral growth. J. Immunol. 130, 2915-2919. Chen, E.Y. and Seeberg, P.H. (1985) Supercoil sequencing: a fast and simple method for sequencing plasmid DNA. DNA 4, 165-171. Couch, R.B., Quarles, J.M., Cate, T.R. and Zahradnik, J.M. (1985) Clinical trials with live cold-reassortant influenza virus vaccines. In: A.P. Kendal and P.A. Patriarca (Eds), UCLA Symposia on Molecular and Cellular Biology, Vol. 36, Options for the Control of Influenza, pp. 223-243. Alan R. Liss, New York. Cox, N.J. and Kendal, A.P. (1984) Genetic stability of A/Ann Arbor/6/60 cold-mutant (temperaturesensitive) live influenza virus genes: analysis by oligonucleotide mapping of recombinant vaccine strains before and after replication in volunteers. J. Infect. Dis. 149, 431-439. Cox, N.J., Kitame, F., Kendal, A.P., Maassab, H.F. and Naeve, C. (1988) Identification of sequence changes in the cold-adapted, live attenuated influenza vaccine strain, A/Ann Arbor/6/60 (H2N2). Virology 167, 554-567. De, B.K., Shaw, M.W., Rota, P.A., Harmon, M.W., Esposito, J.J., Rott, R., Cox, N.J. and Kendal, A.P. (1988) Protection against virulent H5 avian influenza virus infection in chickens by an inactivated vaccine produced with recombinant vaccinia virus. Vaccine 6, 257-261. Edmonson, W.P. Jr., Rothenberg, R., White, P.W. and Gwaltney, J.M. Jr. (1971) A comparison of subcutaneous, nasal and combined influenza vaccination. II. Protection against natural challenge. Am. J. Epidemiol. 93, 480-486. Gwaltney, J.M. Jr., Edmonson, W.P. Jr., Rothenberg, R. and White, P.W. (1971) A comparison of subcutaneous, nasal, and combined influenza vaccination. I. Antigenicity. Am. J. Epidemiol. 93, 472-479. Harmon, M.W., Rota, P.A., Walls, H.H. and Kendal, A.P. (1988) Antibody response in humans to influenza B host-cell derived variants after vaccination with standard (egg-derived) vaccine or natural infection. J. Clin. Microbial. 26, 333-337. Kasel, J.A., Fulk, R.V., Togo, Y., Homick, B.B., Heiner, G.G., Dawkins, A.T. Jr. and Mann, J.J. (1968) Influenza antibody in human respiratory secretions after subcutaneous or respiratory immunization with inactivated virus. Nature (London) 218, 594-595. Kendal, A.P., Maassab, H.F., Alexandrova, G.I. and Ghendon, Y.Z. (1981) Development of cold-adapted recombinant live, attenuated influenza A vaccines in the U.S.A. and U.S.S.R. Antiviral Res. 1, 339-365.
92 Kuroda, K., Hauser, C., Rott, R., Klenk, H.D. and Doerfler, W. (1986). Expression of the influenza virus hemagglutinin in insect cells by a baculovirus vector. Eur. Mol. Biol. Organ. J. 5, 1359-1365. Levitt, N.H., Miller H.V. and Edehnan, R. (1979) Interaction of alphaviruses with human peripheral leukocytes: in vitro replication of Venezuelan equine encephalomyelitis virus in monocyte cultures. Infect. Immun. 24, 642-646. Liew, F.Y., Russell, SM., Appleyard, G., Brand, C.M. and Beale, J. (1984) Cross-protection in mice infected with influenza A virus by the respiratory route is correlated with local IgA antibody rather than serum antibody or cytotoxic T cell reactivity. Eur. J. Immunol. 14, 350-356. Loosli, C.G., Hamre, D. and Berlin, B.S. (1953) Airborne influenza virus A infections in immunized animals. Trans. Assoc. Am. Phys. 66, 222-230. Lowry, O.H., Rosebrough, J.H., Farr, A.L. and Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265-275. Mackett, M. and Smith, G.L. (1986) Vaccinia virus expression vectors. J. Gen. Virol. 67, 2067-2082. Ochiai, H., Kurokawa, M., Hayashi, K. and Niwayama, S. (1988) Antibody-mediated growth of influenza A NWS virus in macrophage-like cell line P388Dl. J. Virol. 62, 20-26. Palese, P. and Schulman, J.J. (1976) Difference in RNA patterns of influenza A virus. J. Virol. 17, 876-884. Ramphal, R., Cogliano, R.C., Shands, J.W. Jr. and Small, P.A. Jr. (1979) Serum antibody prevents murine influenza pneumonia but not influenza tracheitis. Infect. Immun. 25, 992-997. Robertson, J.S., Naeve, C.W., Webster, R.G., Bootman, J.S., Newman, R. and Schild, G.C. (1985) Alteration in the hemagglutinin associated with adaptation of influenza B virus to growth in eggs. Virology 143, 166-174. Rota, P.A., Shaw, M.W. and Kendal, A.P. (1987) Comparison of the immune response to variant influenza type B hemagglutinins expressed in vaccinia virus. Virology 161, 269-275. Rota, P.A., Shaw, M.W. and Kendal, A.P. (1989) Cross-protection against microvariants of influenza virus type B by vaccinia viruses expressing hemagglutinins from egg- or MDCK cell-derived subpopulations of influenza virus type B/England/222/82. J. Gen. Virol. 70, 1533-1537. Salk, J. and Salk, D. (1977) Control of influenza and poliomyelitis with killed virus vaccines. Science 195, 834-847. Schlesinger, J.J. and Brandriss, M.W. (1981) Growth of 17D yellow fever virus in a macrophage-like cell line, U937: role of Fc and viral receptors in antibody-mediated infection. J. Immunol. 127, 659-665. Small, P.A., Smith, G.L. and Moss, B. (1985) Intranasal vaccination with a recombinant vaccinia virus containing influenza hemagglutinin prevents both influenza virus pneumonia and nasal infection: intradermal vaccination prevents only viral pneumonia. In: Vaccine 85, Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases, pp. 175-176. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Smith, G.L., Murphy, B.R. and Moss, B. (1983) Construction and characterization of an infectious vaccinia virus recombinant that expresses the influenza hemagglutinin gene and induces resistance to influenza infection in hamsters. Proc. Natl. Acad. Sci. USA 80, 7155-7159. Stott, E.J., Taylor, G., Ball, L.A., Anderson, K., Young, K.K-Y., King, A.M.Q. and Wertz, G.W. (1987) Immune and histopathological responses in animals vaccinated with recombinant vaccinia viruses the express individual genes of human respiratory syncytial virus. J. Virol. 61, 3855-3861. Tsang, V.C.W., Peralta, J.M. and Simons, A.R. (1983) Enzyme linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. In: J.J. Langone and H.V. Vunakis (Eds), Methods in Enzymology: Immunochemical Techniques, Part E, pp. 377-391. Academic Press, New York. Virelizier, J.L., Allison, A.C. and Schild, G.C. (1979) Immune responses to influenza virus in the mouse, and their role in control of the infection. Br. Med. Bull. 35, 65-68. Waldman, R.H., Bond, J.O., Levitt, L.P., Hartwig, E.C., Prather, E.C., Baratta, R.L., Neill, J.S. and Small, P.A. Jr. (1969) An evaluation of influenza immunization. Influence of route of administration and vaccine strain. Bull. WHO 41, 543-548. Waldman, R.H. and Coggins, W.J. (1972) Influenza immunization: field trial on a university campus. J. Infect. Dis. 126, 242-248.
93 Walls, H., Johansson, K., Harmon, M., Halonen, P. and KendaI, A.P. (1986) Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. J. Chn. Microbial. 24,907-912. Webster, R.G. and Askonas, B.A. (1980) Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice. Eur. J. Immunol. 10, 396-401. Webster, R.G., Reay, P.A. and Laver, W.G. (1988) Protection against lethal influenza with neuraminidase. Virology 164, 230-237. Wenzel, R.P., Hendley, J.O., Sande, M.A. and Gwaltney, J.M. Jr. (1973) Revised (1972-1973) bivalent influenza vaccine: serum and nasal antibody responses to parenteral vaccination. J. Am. Med. Assoc. 226,435-438. Yetter, R.A., Lehrer, S., Ramphal, R. and Small, P.A. Jr. (1980) Outcome of influenza infection: effect of site of initial infection and heterotypic immunity. Infect. Immun. 29, 654-662. Zahradnik, J.M., Kasel, J.A., Martin, R.R., Six, H.R. and Kate, T.R. (1983) Immune responses in serum and respiratory secretions following vaccination with a cold-recombinant (CR35) and inactivated A/USSR/77 (HlNl) influenza virus vaccine. J. Med. Virol. 11, 277-285. (Received 22 September 1989; revision received 11 December 1989)
VIRUS
83
00570
Comparison of inactivated, live and recombinant DNA vaccines against influenza virus in a mouse model Paul A. Rota, Barun K. De, Michael W. Shaw *, Renee A. Black, William C. Gamble and Alan P. Kendal Influenza Branch, Division of Viral and Rickeitsial Diseases, Center for Infectious Diseases, Centers for Disease Control, Public Health Service, Atlanta, GA 30333, U.S.A. (Accepted
13 December
1989)
Summary
The protective efficacy of influenza hemagglutinin expressed from recombinant vaccinia virus was compared with that induced by inactivated or infectious influenza vaccines. Intraperitoneal and intranasal routes of vaccination were compared. All the vaccines except the intranasally administered, inactivated vaccine induced detectable levels of neutralizing and hemagglutination-inhibiting antibodies in the serum of mice at 28 days postvaccination. Immunization with any of the intranasally administered vaccines reduced the amount of influenza virus nucleoprotein antigen in lungs after challenge with a homologous, mouse-adapted strain of influenza virus. Intraperitoneally administered vaccines failed to provide such protection. These results indicated that the route of vaccine administration may be the most critical factor for inducing protective immunity. The results also showed that in this mouse model a recombinant DNA-based vaccine could provide protection equivalent to that provided by conventional attenuated and inactivated influenza vaccines. Influenza virus; DNA vaccine; Mouse model
Correspondence ro: P.A. Rota, Influenza Branch, G-16, Centers for Disease Control, Atlanta, GA 30333, U.S.A. * Present address: M.W. Shaw, Department of Epidemiology. University of Michigan School of Public Health, AM Arbor, MI 48109, U.S.A. 0168-1702/90/$03.50
0 1990 Elsevier Science Publishers
B.V. (Biomedical
Division)
84
Introduction Several approaches have been evaluated in an attempt to develop improved vaccines against influenza viruses, an effort complicated by the continual antigenic variation of the virus. Currently only inactivated virus vaccines composed of purified whole virus, chemically disrupted virions or purified subunit components have been licensed for use in the United States. Live attenuated vaccines have been widely used in a few countries and are undergoing clinical evaluation in the United States (Kendal et al., 1981; Couch et al., 1985). In theory, subunit vaccines could be prepared by using recombinant DNA expression systems. For example, human influenza virus hemagglutinin (HA) has been expressed from recombinant vaccinia virus and been shown capable of eliciting hemagglutination-inhibiting (HI) antibody in rodents. These animals were partially (Smith et al., 1983) or fully protected (Small et al., 1985) after challenge with homologous strains of influenza virus. Noninfectious vaccines containing the HA (Kuroda et al., 1986; De et al., 1988) or infectious vaccines containing the neuraminidase (Webster et al., 1988) of avian influenza viruses that were derived from baculovirus or vaccinia virus expression vectors have protected chickens against lethal challenge with virulent avian influenza viruses. However, no studies have yet been reported that directly compared recombinant DNA-derived influenza vaccines with traditional inactivated or live attenuated vaccines. Additionally, the route of administration has been suggested to be an important determinant in the efficacy of influenza vaccines (e.g. Small et al., 1985). While vaccination by the parenteral route is best for the induction of a humoral response, vaccination in the upper respiratory tract may be preferred for inducing a protective local antibody response against a respiratory virus (Liew et al., 1984). Therefore, we have compared the relative protection seen in a mouse model system after vaccination by the intranasal or parenteral routes with inactivated, live, and recombinant DNA-derived vaccines containing the same influenza type A hemagglutinin. To evaluate vaccine efficacy, we have measured both serum neutralizing antibody and virus production in the affected organs after challenge with a mouse-adapted strain of influenza virus.
Materials and Methods Viruses and vaccines Influenza viruses were cultivated in embryonated hen’s eggs. Influenza A/Ann Arbor/6/60 (H2N2) wild-type (WT) virus was from the stocks of the World Health Organization Influenza Center (WHOIC) at the Centers for Disease Control (CDC) in Atlanta, Georgia (originally obtained from Dr H.F. Maassab, University of Michigan). Two mutants of this virus, cold-adapted (CA) A/Ann Arbor/6/60 and mouse-adapted pathogenic (MA) A/Ann Arbor/6/60 were also kindly provided by Dr Maassab. The MA virus had been passaged in mouse lungs 44 times before being
85
amplified in the allantoic cavity of emb~onated hens’ eggs prior to use in subsequent experiments. The infectivity of the final egg-derived MA virus was assayed in mice before the challenge experiments. The purification of viruses and virion RNA were as previously described (Palese and Schulman, 1976; Cox and Kendal, 1984). Sequencing Sequences of the influenza HA genes were obtained from viral RNA by using a modification of the Sanger dideoxy method (Chen and Seeberg, 1985; Robertson et al., 1985). Primers used in the sequencing reactions have been described by Cox et al. (1988).
Vaccines We constructed recombinant vaccinia viruses expressing the HA of A/Ann Arbor/6/60 by using previously described techniques (Mackett and Smith, 1986; Rota et al., 1987; De et al., 1988). The expression of authentic influenza HA was verified by Western blot analysis of lysates prepared from cells infected with the recombinant virus (Tsang et al., 1983) by using rabbit hyperimmune antiserum against whole A/Ann Arbor/6/60 virions. Cell surface localization of the HA expressed from the recombinant vaccinia virus was also verified by immunoelectron microscopy (De, unpublished observations). Recombinant vaccinia virus vaccine was purified and titrated by plaque assay as previously described (De et al., 1988). For preparation of inactivated influenza vaccine, ~adient-puffin A/Arm Arbor,/6/60 WT virus was resuspended in phosphate-buffered saline (PBS) containing 0.15% formalin. After a 48 h incubation at 4”C, the virus was pelleted from the formalin-containing buffer (100,000 x g, 1 h, 4°C) and resuspended in PBS. Inactivation was monitored by titrating infectivity in MDCK cell cultures. No plaque-forming units were detected in standard plaque assays of the inactivated vaccine preparation at any dilution up to concentrations causing cytopathic effect solely from the input inoculum. Commercially produced inactivated influenza vaccines were used as negative controls in these infectivity assays. Protein concentration was determined by using the Lowry assay (Lowry et al., 1951).
Animal ~~ccinati~~ and ~~a~len~eexperiments Vaccines were administered to female A/J mice 5-7 weeks old by either the intranasal or the intraperitoneal route. Intrauasal vaccines were given in a lo-p1 volume under CO, anesthesia. Intraperitoneal vaccines were given in a volume of 0.5 ml. Serum samples for antibody titration were obtained by sacrificing groups of five mice and exsanguinating them at intervals after vaccination. The concentration of neutralizing antibody pooled serum samples was determined by using the microneutralization assay described by Harmon et al. (1988). Mice were challenged with the MA A/Arm Arbor/6/60 virus (FlM44E3). Since the level of anesthesia has been shown to effect the outcome of influenza infection
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(Yetter et al., 1980), mice were individually anesthetized with CO, and infected intranasally with approximately 1 X lo4 mouse ID,, of the MA virus in a volume of 10 ~1. Lung homogenates were prepared from groups of five mice at 2, 3,4, 5,8, and 10 days postchallenge as described previously (Rota et al., 1987). The concentration of influenza type A nucleoprotein (NP) antigen present in the pooled lung homogenates was determined by using time-resolved fluoroimmunoassay (TR-FIA) (Walls et al., 1986). All samples were run in triplicate and the results were corrected for lung weights.
Results
HA sequence changes associated with mouse adaptation
Because it is known that laboratory passage can select for different subpopulations of influenza virus, the sequences of the A/Ann Arbor/6/60 HAS in the vaccine and challenge viruses were compared by direct sequencing of viral RNA. A total of 11 changes was noted when the deduced amino acid sequences of the HA genes of the WT, CA, and MA A/Ann Arbor/6/60 viruses were compared (Table 1). Of these, nine were in the HA1 polypeptide. None occurred in the known major antigenic sites. Changes at five positions (53, 71, 180, 221, and 299) would cause significant changes in local hydropathy. Also, the changes at positions 180 and 221 occur in an area close to the receptor-binding pocket of the HA. However, as presented in the following, these differences did not seem to interfere with the generation of protective immune responses in test animals.
TABLE 1 Changes in the amino acid sequences of the hemagglutinins of the cold-adapted and mouse-adapted influenza A/Ann Arbor/6/60 viruses Position
Wild type
CA’
MA2
18 53 71 151 180 199 221 264 299 367 378
Asn
Be
Asp Ser
Asp Ser
Ile Val
LYS Ile
LYS Asn
Gly A% LYS Glu
G’Y
Lys Ala
’ Cold-adapted. 2 Mouse-adapted (pathogenic).
GlY LYS Gly LYS Ser
Arg Asn Ile Val A% Gln Glu A% Ser
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Response to vaccination To determine the optimum dose for each vaccine, we inoculated mice with serial dilutions of each of the experimental influenza vaccines. Mice to be used in challenge studies were given a dose of vaccine equivalent to 50 times the highest dilution that had induced detectable serum neutralizing antibodies. However, for the inactivated, intranasal vaccine, the dose given was the same as that used for intraperitoneal vaccination. The doses used and the levels of neutralizing antibodies induced at 30 days postvaccination are shown in Table 2. All the vaccines induced detectable levels of neutralizing antibodies in serum except for the intranasally administered, formalin-inactivated influenza virus.
Protection studies A/J mice were selected for our study because, of four inbred strains tested, they were the most susceptible to infection with the particular mouse-adapted influenza A virus we were using (results not shown). Groups of mice immunized as described earlier were challenged intranasally with MA A/Ann Arbor/6/60 virus 30 days after vaccination. Protection was evaluated by measuring with TR-FIA the reduction in yield of influenza A virus NP antigen in the lungs at various times after challenge (Fig. 1). Results from this method have been found to correlate well with results from plaque assays on MDCK cell monolayers (Rota et al., 1987, and unpublished results). The controls for each challenge experiment consisted of mice vaccinated with PBS or with WT vaccinia virus (New York Board of Health strain). Initially, vaccination with live CA A/Ann Arbor/6/60 virus vaccine or live WT (not mouse-adapted) virus given intranasally was compared to intraperitoneal vaccination with recombinant vaccinia virus expressing the H2 HA or inactivated influenza virus vaccines (Fig. 1).
TABLE
2
Experimental
influenza
type A (H2N2)
vaccines
Vaccine
Dose/mouse
Route
Live vaccines A/Ann Arbor/6/60 (WT) A/Ann Arbor/6/60 (CA) vaccinia A/AA-HZHA vaccinia A/AA-HZHA
2.5 5.0 4.0 1.0
IN IN IP IN
800 200 200 100
Formalin-inactivated A/Arm Arbor/6/60 A/Ann Arbor/6/60
1.3 pg HA 1.2 pg HA
IP IN
100 < 25
’ Serum neutralizing
vaccines (WT) (WT)
x x x x
lo6 10’ lo5 lo6
EID,, EIDs, PFU PFU
Titer ’
antibody titer vs. A/Ann Arbor/6/60 (wt) at 30 days post-vaccination. average of three replicates of each serum pool (Abbreviations: PFU, plaque-forming intranasal; IP, intraperitoneal; EID, egg infectious dose).
Number is units; IN,
88
6.5 6.25
5.75
6
5.5
5.75 5.5
5.25
5.25
5
5
4.75
4.75 4.5
4.5
4.25
4.25
4
4
3.75 3.5
3.75
3.25
3.5
3
3.25
2.75 , 0
, 1
, 2
, 3
, 4
, 5
, 6
, 7
, 8
, 9
1
10
’
2.5 d
1
i.
3
4
5
6
7
8
9
10
Fig. 1. Comparison of vaccination with recombinant vaccinia virus or inactivated influenza virus by the intraperitoneal route and vaccination with live influenza virus by the intranasal route. Mice were vaccinated with formalin-inactivated (filled triangle), CA (asterisk), WT (open square) A/Ann Arbor/6/60 virus or recombinant vaccinia virus expressing the H2 HA (X), or PBS (filled square). PBS, formalin-inactivated and recombinant vaccinia virus were given intraperitoneally; WT and CA viruses were given intranasally. Figure shows the amount of influenza NP antigen (as log,, of TR-FIA counts per minute (cpm)) in lung homogenates (y axis) at 0 through 10 days postchallenge with MA virus (x axis). Mice were challenged 30 days (Panel A) or 3 months (Panel B) after initial vaccination.
The levels of influenza NP antigen detected in the control groups after challenge were nearly identical at all the time points tested. Because vaccinia virus alone did not induce non-specific antiviral activity in the mice only the antigen levels for the PBS-vaccinated control mice are shown. In these control mice, NP antigen was detected at 3 days postinfection and peaked at 4-6 days postinfection; antigen levels returned to background levels by 8 days postinfection. Mice receiving any of the vaccines except the intranasally administered inactivated virus had neutralizing antibodies in their serum at the time of challenge (Table 2). However, only the intranasally administered, live influenza viruses significantly reduced the amount of influenza virus NP antigen detected in the lung homogenates after challenge (Fig. lA,B). Protection from these intranasal vaccines was nearly complete; antigen levels rose only slightly above background readings at day 0, and all groups had at least a 97% reduction in detectable antigen compared with the peak levels detected in control animals. Similar levels of antigen reduction were observed when vaccinated mice were challenged at 3 months postvaccination (Fig. 1B). In contrast, the single dose of formalin-inactivated vaccine given intraperitoneally failed to reduce virus antigen levels in lung homogenates, and peak antigen levels in these animals were as high as or higher than in the control animals whether challenge was 1 or 3 months after vaccination (Fig. lA,B). In addition, an increase in antigen was consistently detected earlier in these animals than in controls. An intermediate level of protection was produced by intraperitoneal inoculation of the recombinant vaccinia virus expressing the H2 HA. Although a rise in NP
89
..._ \
i
4.5 / I
4.25
\
3.25
3’
, 0
, 1
, 2
, 3
, 4
, 5
, 6
, 7
, 8
(
9
,
’
IO
Fig. 2. Results of vaccination by the intranasal route. Figure shows the amount of influenza nucleoprotein [as log,, of TR-FIA counts per minute (cpm)] in lung homogenates at 0 through 10 days after challenge with MA virus. Mice were challenged at 30 days postvaccination. Symbol assignments are the same as those for Fig. 1 except that the formalin-inactivated and recombinant vaccinia virus vaccines were given intranasally.
antigen was detected, peak antigen levels in mice vaccinated with this recombinant vaccinia virus were lower than peak antigen levels in control animals whether challenged at 1 or at 3 months postvaccination (Fig. lA,B). To test the effect of the route of administration on the ability of the vaccines to protect against influenza challenge, mice were vaccinated with the formalin-inactivated and the recombinant vaccinia virus vaccines by the intranasal route before challenge (Fig. 2). Only mice given the recombinant vaccinia virus developed detectable serum neutralizing antibodies when vaccinated by this route (Table 2). However, when either group of mice was challenged with the MA A/Ann Arbor virus, both were highly protected. This protection, though not complete, was nearly equivalent to the protection provided by the intranasally administered live WT A/Ann Arbor influenza virus. All the vaccinated mice had at least a 97% reduction in the amount of peak antigen levels compared to control mice.
Discussion The primary purpose of this investigation was to expand on earlier studies that had examined the efficacy of vaccination with recombinant vaccinia viruses expressing HA. It had been shown that recombinant vaccinia viruses expressing the H3 HA provided partial protection when given parenterally (Smith et al., 1983) and full protection when administered intranasally (Small et al., 1985). In contrast, parenterally administered recombinant vaccinia virus expressing the influenza B HA gave full protection against challenge with a non-mouse-adapted strain of influenza B (Rota et al., 1987,1989). A study by Stott and coworkers (1987) showed that mice vaccinated with vaccinia recombinants expressing RSV G protein developed equiv-
90
alent levels of serum antibody after intranasal or intraperitoneal vaccination. However, the levels of antibody in the lungs of the intranasally vaccinated mice were more than lo-fold greater than in mice vaccinated by the intraperitoneal route. The results of the challenge experiments presented here confirmed that the route of administration was more important for the induction of protective immunity than the actual composition of the vaccine. Presumably, only low levels of local antibody were induced in the respiratory tract by intraperitoneal vaccination, and these levels were insufficient to provide complete protection against the mouse-adapted influenza virus used for the challenge in spite of the presence of neutralizing antibody in the serum. The role of local immunity in the upper respiratory tract is emphasized by the response of mice vaccinated with intranasally administered, inactivated vaccine: serum antibody was essentially undetectable prior to challenge, yet the animals were protected. Results of challenge experiments may differ according to the degree of adaptation or pathogenicity of the challenge virus. The measures of virus antigen depicted in Figs. 1 and 2 include only the lower respiratory tract; the entire lung, trachea and bronchi were excised and assayed. It may be that the apparent unrestricted growth of the virus after challenge of intraperitoneally immunized mice was limited to the trachea, and the mice were protected against a potentially fatal pneumonia. It has been clearly shown in the mouse system that serum antibody prevents viral pneumonia but not tracheitis or rhinitis (Loosli et al., 1953; Ramphal et al., 1979; Small et al., 1985). In our study, all the vaccinated animals survived, whereas lo-30% of the control animals died, depending on the experiment. These levels of viral pathogenicity and vaccine protection may be more relevant to human influenza than are the levels of pathogenicity and protection observed in model systems where highly lethal influenza viruses are used for challenge. At this time, we have no explanation for the finding that the peak levels of influenza antigen detected in the lungs after challenge were greater in mice that had been vaccinated by the intraperitoneal route than in the unimmunized control animals. Antibody enhancement of viral infectivity may be considered. Enhanced uptake by macrophage-like cells of antibody-coated influenza virus has been implicated in some other systems, possibly through Fc receptor-mediated phagocytosis (Webster and Askonas, 1980; Ochiai et al., 1988). This phenomenon has also been observed after infection with other viruses such as Venezuelan equine encephalitis virus (Levitt et al., 1979), yellow fever virus (Schlesinger and Brandriss, 1981), and reovirus (Burstin et al., 1983). Most of the influenza studies have shown a direct inverse correlation between influenza vaccine-induced serum antibody levels and disease incidence (reviewed in Salk and Salk, 1977). The roles of local humoral and cellular immunity are not fully understood and may differ when vaccines are administered intranasally or parenterally. It has been reported that nonreplicating virus administered intranasally was capable of inducing local immunity (Kasel et al., 1968). Later studies on the effects of intranasal, inactivated influenza vaccines showed greater protection from aerosolized vaccine than from the same vaccine given subcutaneously (Waldman et al., 1969; Waldman and Coggins, 1972; Zahradnik et al., 1983). However, other studies
91
on humans have failed to confirm the advantage of local over parenteral immunization with inactivated vaccine (Gwaltney et al., 1971; Edmonson et al., 1971; Wenzel et al., 1973). Despite the many potential drawbacks pertaining to the use of artificial models for human influenza, the observations reported here suggest a need for further studies of vaccination by the intranasal route. These studies should include inactivated vaccines and live attenuated virus as well as antigens produced by recombinant expression systems.
Acknowledgement
The authors are grateful to Jennifer Rota for assistance with the TR-FIA assays.
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93 Walls, H., Johansson, K., Harmon, M., Halonen, P. and KendaI, A.P. (1986) Time-resolved fluoroimmunoassay with monoclonal antibodies for rapid diagnosis of influenza infections. J. Chn. Microbial. 24,907-912. Webster, R.G. and Askonas, B.A. (1980) Cross-protection and cross-reactive cytotoxic T cells induced by influenza virus vaccines in mice. Eur. J. Immunol. 10, 396-401. Webster, R.G., Reay, P.A. and Laver, W.G. (1988) Protection against lethal influenza with neuraminidase. Virology 164, 230-237. Wenzel, R.P., Hendley, J.O., Sande, M.A. and Gwaltney, J.M. Jr. (1973) Revised (1972-1973) bivalent influenza vaccine: serum and nasal antibody responses to parenteral vaccination. J. Am. Med. Assoc. 226,435-438. Yetter, R.A., Lehrer, S., Ramphal, R. and Small, P.A. Jr. (1980) Outcome of influenza infection: effect of site of initial infection and heterotypic immunity. Infect. Immun. 29, 654-662. Zahradnik, J.M., Kasel, J.A., Martin, R.R., Six, H.R. and Kate, T.R. (1983) Immune responses in serum and respiratory secretions following vaccination with a cold-recombinant (CR35) and inactivated A/USSR/77 (HlNl) influenza virus vaccine. J. Med. Virol. 11, 277-285. (Received 22 September 1989; revision received 11 December 1989)
