Immunogenicity and pathogenicity of temperature-sensitive modified respiratory syncytial virus in...

Journal of Medical Virology 25:411-421 (1988)

lmmunogenicity and Pathogenicity of Temperature-Sensitive Modified Respiratory Syncytial Virus in Adult Volunteers Elizabeth McKay, Peter Higgins, David Tyrrell, and Craig Pringle

MRC Virology Unit, Institute of Virology, Glasgow, Scotland (E. M.), MRC Common Cold Unit, Harvard Hospital, Salisbury (P. H., D. T.), and Biological Sciences Department, University of Warwick, Coventry (C. P.), England

Single temperature-sensitive (ts) mutants of a subgroup A strain of respiratory syncytial (RS) virus whose multiplication is restricted at 39°C in MRC-5 cells and double ts mutants that are restricted at 38"C, were obtained following mutagenesis using 5-fluorouracil and acridine-like compounds. Isolation and propagation of the parental RSS-2 strain of RS virus and its derived ts mutants were carried out entirely in MRC-5 human diploid cells.

The immunogenicity and disease-producing ability of four of these mutants and the parental unmodified strain have been assessed by intranasal administration into groups of about 20 adult volunteers. The results of these trials indicate that the capacity of the parental RSS-2 strain to produce upper respiratory tract infection in adults was not diminished by limited propagation in MRC-5 cells. The mutants on the other hand were impaired in this respect to varying extents. The double mutant tslB in particular has characteristics that suggest that it may be suitable for further development as a live RS virus vaccine. It retained near normal immunogenicity and replicative ability in the upper respiratory tract, while exhibiting greatly reduced disease-producing potential.

Key words: respiratory syncytial virus, temperature-sensitive mutants, vaccine, adult volunteers

INTRODUCTION

Respiratory syncytial (RS) virus is responsible for most of the serious respiratory illness experienced in infancy. RS virus is associated particularly with bronchiolitis and pneumonia in the first months of life, and epidemics occur annually during the winter in temperate climates and during the rainy season in tropical and subtropical regions. Reinfection causing upper respiratory disease can occur throughout life and

Accepted for publication February 24, 1988

Address reprint requests to Craig Pringle, Biological Sciences Department, University of Warwick, Coventry CV4 AL, UK.

0 1988 Alan R. Liss, Inc.

412 McKay et al.

may again become life threatening in the elderly. Successful immunoprophylaxis of RS virus infection would have a major impact on child health and might also reduce some of the respiratory illness experienced by adults and the elderly.

Early attempts to control RS virus infection using inactivated vaccine were unsuccessful and indeed led to exacerbation of disease on subsequent exposure of vaccinees to natural infection [Chin et al., 1969; Kim et al., 19691. Although measles and mumps, which are also diseases caused by paramyxoviruses, can be successfully controlled by parenterally administered live attenuated vaccines, no comparable live RS virus vaccine is available. Parenteral administration of an essentially unmodified live RS virus [Bunyak et al., 1978, 19791 induced a modest immune response but did not reduce the frequency of upper and lower respiratory tract disease following subsequent naturally acquired RS virus infection [Belshe et al., 19821, perhaps because viraemia is absent or negligible in RS virus infection. Nonetheless, natural infection with RS virus confers a degree of immunity, and high titres of serum antibody are stimulated. The efficacy of a live vaccine may depend on the amount of serum antibody or perhaps secretory antibody induced. Several strains of RS virus modified for growth at the lower temperature of the upper respiratory tract were developed, but none has proved clinically acceptable owing either to genetic instability or overattenuation. A low-temperature (26°C) grown live vaccine derived from the A2 strain of RS virus exhibited reduced virulence in adult volunteers [Friedewald et al., 19681. The same vaccine produced a silent infection in young children who had been exposed to natural infection with RS virus, but produced mild lower respiratory tract disease in infants who had not been infected with RS virus previously [Kim et al., 197 11. The partial success of this vaccine encouraged the development of temperature-sensitive (ts) mutant vaccines from the A2 wild-type strain, the rationale being to derive a temperature-restricted variant that would multiply only in the lower- temperature environment of the upper respiratory tract following intranasal administration and induce local protective IgA secretory immunity. Mutant tsl, which did not multiply in vitro at 37°C or produce disease when administered intranasally to adult volunteers, did induce resistance to subsequent challenge with wild-type virus. There was no indication of reversion [Wright et al., 19711. In infants without prior experience of RS virus, however, the vaccine induced mild disease and underwent reversion when moderate doses (104-105 TCIDSO) were administered [Kim et al., 1973; Hodes et al., 19741. Attempts to increase the temperature restriction of this vaccine resulted in overattenuation [Richardson et al., 19771. Mutant ts2, another temperature-sensitive candidate vaccine derived from the A2 strain, which as well as being restricted for growth at 37°C produces nonsyncytial plaques in cultured cells indicative of a defective fusion protein, was shown to be highly attenuated in the chimpanzee. However, it failed to infect the majority of seronegative infants even at high doses ( lo6 TCID~O) [Wright et al., 19821.

These A2 strain vaccines were developed and propagated in primary or secondary cultures of bovine kidney cells and hence were vulnerable to contamination by extraneous agents and perhaps also to the risk of progressive deadaptation for growth in human cells. The present report describes the modification of a more recent isolate of RS virus carried out entirely in the MRC-5 human foetal lung diploid cell line. Ts mutations were induced sequentially using two mutagens with different modes of action to obtain greater inherent genetic stability and to limit replication to the cells of the upper respiratory tract. The immunogenicity and disease-producing potential

Temperature-Sensitive Mutants of RS Virus 413

of four of these variants, two single and two double mutants, have been assessed by intranasal administration to adult volunteers. The results of these trials, which are summarised in this paper, suggest that one of these modified strains has characteristics that make it suitable for further development as a potential live RS virus vaccine. A more detailed analysis of the immune response to the individual proteins of the virus will be published separately (Watt et al., unpublished data).

MATERIALS AND METHODS Cell Culture

Isolation and propagation of virus were carried out entirely in the MRC-5 line of human diploid cells, which is an approved substrate for vaccine development. An early-passage culture of MRC-5 cells was obtained from Dr. Jacobs of the National Institute of Biological Standards and Control, Hampstead. The cultures propagated from this material were routinely checked for freedom from mycoplasmal contamination by fluorescent staining [Russell et al., 19751. The cells were maintained in Eagle's medium (Glasgow modification) supplemented with antibiotics ( 100 unitdm1 penicillin, 100 pg/ml streptomycin) and 5 % heat-treated foetal calf serum (Gibco- Biocult).

Media and Chemicals

5-Fluorouracil was supplied by Sigma Limited (London); the acridine-like compounds ICR 372 and ICR 340 synthesized at the Institute of Cancer Research, Philadelphia were obtained from Dr. Georgine Faulkner (Biological Sciences Depart- ment, University of Warwick) who had shown that these compounds were mutagenic for bovine rotaviruses [Faulkner-Valle et al., 19821. Eagle's medium was prepared in the Media Preparation Department of the Institute of Virology.

Infectivity Assay

Infectivity assays were carried out using monolayers of MRC-5 cells. First, lo6 MRC-5 cells were seeded into 30-mm plastic Petri dishes and inoculated when just confluent, usually 24-48 hours after seeding; 0.2 ml was inoculated per dish, and the inoculum was absorbed for 30 minutes at 33°C. The infected monolayers were incubated under 0.9% Bacto agar (Difco) containing overlay for 7 days at 33°C or the appropriate restrictive temperature (37"C, 38"C, or 39°C) in a C02-gassed incubator. The monolayers were fixed in 1 % glutaraldehyde and stained with Giemsa stain.

lmmunofluorescence

as described previously [Faulkner et al., 19761.

Radiolabelling, Extraction of Labelled Polypeptides, and Sodium Dodecyl Sulphate (SDS)/Polyacrylamide Gel Electrophoresis

Radiolabelling with [35S]-methionine and analysis of viral protein synthesis by SDS-polyacrylamide gel electrophoresis were carried out by methods described previously [Pringle et al., 19781.

Examination of cells by indirect immunofluorescence technique was carried out

414 McKay et al.

Antibody Assay

Antibodies to RSV were detected by an indirect enzyme-linked immunosorbent assay (ELISA) test. Suitably diluted serum samples were added to plates coated with a tissue culture fluid antigen of RS virus. The subsequent addition of alkaline phosphatase conjugated to antihuman IgG or IgA followed by the substrate p-nitro- phenol phosphate resulted in a change of colour when antibody was present in the serum. The optical densities (OD) of serum samples at three different dilutions, 1 :250, 1 :500, and 1 : 1,000, were measured, and the highest pre/postchallenge OD ratio at these dilutions was recorded as the ER (ELISA ratio). An ER greater than the mean ratio + 3 SD of serum from volunteers given saline instead of RS virus was considered to be a significant antibody rise. An assessment of the antibody status of the volunteers prior to challenge was made by measuring the OD of the preserum diluted 1 :250.

Clinical Assessment of Volunteers

Volunteers were observed daily by a clinician who was unaware of the type of virus challenge used. All signs and symptoms together with the number of tissues used were recorded and scored according to a standard protocol [Beare and Reed, 19771. The weight of nasal secretion produced each day was also measured. At the end of the trial a clinical judgement was made for each volunteer as to whether they had suffered a cold. Colds were graded as doubtful (symptoms not sufficiently characteristic or persistent to allow for firm diagnosis), mild, moderate or severe.

These studies received prior approval from the Harrow District Ethical Committee.

Isolation of the RSS-2 Strain

An isolate of RS virus was required as the starting material, which had not been passaged through heteroploid or animal cells and which, preferably, was more rele- vant to the disease situation in Britain than the laboratory strains available at the time, all of which had been isolated either in North America or Australia. With the assistance of Professor Phillip Gardner, two strains of virus designated RSS-1 and RSS-2 were isolated by inoculating nasopharyngeal aspirates from cases occurring at the height of the winter RS virus epidemic in 1976 directly into MRC-5 cell cultures at the Royal Victoria Hospital, Newcastle-upon-Tyne. These viruses were isolated from twin infants experiencing bronchiolitis of sufficient severity to require hospital care. The two strains were indistinguishable by any criteria then available, and one of them, the RSS-2 strain, was chosen arbitrarily as the parental wild-type virus. Subsequently, when it became apparent that two distinct antigenic subgroups of RS virus were prevalent in the human population [Anderson et al., 19851, it was shown that the RSS-2 strain belonged to the A subgroup of RS virus [Gimenez et al., 19861 E.J. Stott, personal communication]. The RSS-2 strain used by us and the A2 strain used by Chanock and his colleagues for vaccine development both belong to subgroup A, and despite having been isolated in different continents 15 years apart show considerable homology at the nucleotide level [Baybutt and Pringle, 19871.

The inoculation of the MRC-5 cell cultures was carried out in the paediatric ward, and the inoculated cultures were immediately transferred to a laboratory reserved solely for propagation of this virus at the MRC Common Cold Unit, Salisbury. A genetically homogeneous stock was established by three sequential


isolations from individual plaques on MRC-5 monolayers inoculated with limiting dilutions of the virus. All subsequent work was carried out in a self-contained laboratory unit at Ruchill Hospital, Glasgow, devoted entirely to the modification of this virus and the maintenance of the MRC-5 cells used for the routine production of monolayer cultures. The assay of infectious RS virus in nasal aspirates from volunteers was undertaken at the University of Warwick using BS-C-I cell monolayers.

RESULTS Isolation of ts Mutants: First-Stage Mutagenisation

The wild-type RSS-2 strain virus was mutagenised by growth in the presence of concentrations of 5-fluorouracil in the range 50-200 pg/ml. Released virus was plated out under agar overlay, and well-spaced plaques from single or low-count plates were picked directly into fresh cell cultures in small screw-capped flasks. Continuous propagation without intervening freeze-thawing proved to be an essential factor in successful isolation of ts mutants, since no mutants were obtained among several hundred clones isolated from mutagenised virus stored at - 70°C. Therefore these primary cultures were immediately screened for plaque formation on MRC-5 cell monolayers incubated at 33°C and 39°C.

Virus stocks that failed to produce plaques at 39°C were recloned from single plaques at limiting dilution and retested. Stocks of each confirmed ts mutant were then prepared. A total of 20 ts mutants, which exhibited an efficiency of plating of <0.001% at the restrictive temperature of 39°C relative to the permissive temperature of 33"C, were obtained from 1,450 plaque isolates. The frequency of ts mutants (1.3%) isolated from mutagenised virus compared with a frequency of <0.58% spontaneous ts mutants present in unmutagenised virus indicated that the mutants obtained were indeed induced by the mutagenic treatment.

The mutant stocks were examined by direct immunofluorescence for ability to induce synthesis of intracellular viral antigens at 39"C, and for induction of virus- specific protein synthesis at 39°C by SDS/polyacrylamide gel electrophoresis of cell extracts (data not shown). Two mutants with contrasting properties were chosen as substrates for second-stage mutagenesis. Mutant ts 19(A) was unable to induce detect- able antigen or virus protein synthesis in BS-C-1 cells at 39°C. Mutant tsl(A), on the other hand, was able to induce near-normal levels of immunofluorescence and virus protein synthesis in infected BS-C-1 cells at 39°C. (The letter A in parentheses is used to distinguish the mutant derived in the first mutagenesis from that derived in the second mutagenesis (B)).

Isolation of ts Mutants: Second-Stage Mutagenesis

Monolayers of MRC-5 cells were inoculated at low multiplicity with mutants tsl(A) and ts19(A). After adsorption at 33°C for 1 hour, the inoculum was removed, and the monolayers were washed before addition of incubation media containing the mutagens ICR 340 or ICR 372 at concentrations of 1, 2, 3, 5 , or 10 pg/ml. These cultures were incubated in the dark as described by Faulkner-Valle et al. [ 19821 until the cytopathic effect was extensive. These mutagenised stocks were plated, without intervening freeze-thawing, onto monolayers of just-confluent MRC-5 cells and over- layed with medium containing 0.9% agar and 2% foetal calf serum. Plaques were isolated from neutral-red-stained plates after 5 days incubation at 33"C, and mini-

416 McKay et al.

stocks were established by a single passage in small culture flasks containing approximately lo6 MRC-5 cells.

These stocks were then assayed for plaque formation on monolayers of MRC-5 cells incubated at 33°C and 38°C. In the case of tsl9(A), three out of 253 clones (1.19%) isolated from ICR 340-mutagenised virus failed to produce plaques at 38"C, and four out of 220 clones ( 1.8 1 %) from ICR 372 mutagenised virus failed to produce plaques. Five of 372 clones (1.34%) obtained from mutant tsl(A) mutagenised using ICR 340 similarly failed to form plaques at 38°C.

These frequencies of isolation of mutants are well above the level of spontaneous ts mutants present in these stocks confirming that the mutants were probably induced by exposure of the virus to these acridine-like compounds during replication. Individ- ual mutants from the ICR 340-mutagenised virus, designated tsl(B) and tsl9(B), respectively, to indicate their origin by two stages of mutagenesis, were recloned by isolation from a single plaque, and stocks were prepared for characterisation. The plaque-forming properties according to temperature of tsl(A), tsl(B), ts19(A), ts19(B), and wild-type virus are given in Table I.

The nature of the mutational lesions has not been determined. Mutant ts19(A), however, behaved as a RNA-negative mutant and did not synthesize virus-specific polypeptides at 39°C. Mutant tsl(A), on the other hand, induced near-normal levels of polypeptides at 39°C and therefore has an RNA-positive phenotype. The phenotype of the B series mutants as regards intracellular polypeptide synthesis corresponds to that of the A series, except that the temperature threshold of restriction has been reduced by approximately 1 "C (data not shown).

Evaulation of First- and Second-Stage ts Mutants in Adult Volunteers

As a first stage in assessment of the extent of modification of the disease- producing potential of these mutants, each of the four mutants and the wild-type virus were administered intranasally to groups of between 20 and 22 healthy adult volunteers as part of the regular research programme of the MRC Common Cold Unit, Salisbury. The wild-type virus administered to the volunteers had undergone approx-

TABLE I. Assay of the Temperature Sensitivity of the tslA, tslB, tsl9A, tsl9B, and RSS-2 Wild- Type Stocks on BS-C-1 and MRC-5 Cells*

Assay Temperature of incubation Virus system 33°C 37°C 38°C 39°C

RSS-2 Wild BS-C-1 1.4 x 105 1.6 x 105 1.6 x 105 1.8 x lo5 TY Pe

MRC-5 4 x lo4 8.5 x lo4 3.5 x lo4 4.3 x lo4

fs 1 A BS-C- 1 2.1 x lo5 N.D. 2 x lo4 Nil MRC-5 1.3 x lo4 N.D. 1.3 x lo4 Nil

rs lB BS-C-1 5 x lo5 2.3 x 105 1.2 x 102 Nil MRC-5 4 x lo4 2.0 x lo3 Nil Nil

r s 19A BS-C- 1 1.4 x 105 N.D. 5 x 10' Nil MRC-5 2.3 x lo4 N.D. 9.5 x 102 Nil

rs19B BS-C- 1 4.4 x lo5 6 x lo3 Nil Nil MRC-5 5.5 x lo4 6 x lo3 Nil Nil

*The figures in the table are plaque forming units per ml.; N.D. = no data; Nil = no plaques detected (< 5 ph/rnl).


imately the same aggregate number of passages as required for production of the ts mutant derivatives. The volunteers, male or female, were aged between 18 and 50 years. They were housed in isolation at the Unit in flats for two to three persons for 10 days. After a quarantine period of 3 days those that were clinically normal and that had normal chest X-ray, blood count and urine examination were allocated at random to receive intranasally 1 ml of tissue culture fluid containing approximately 1,000 plaque-forming units (pfu) of virus. Any volunteer showing evidence of intercurrent infection as a quarantine cold, or by retrospective analysis, was excluded from the trial. None of the volunteers was seronegative, since RS virus infection occurs early in life, and the virus is ubiquitous. The volunteers were observed according to the standard protocol of the Unit (see “Materials and Methods”). Daily nasal washings were collected and immediately frozen at -70°C for subsequent assay for the presence of released infectious virus. The samples were not decoded until completion of the experiment. Serum samples were collected on arrival in the Unit and at about 21 days after inoculation. The samples were examined later for specific antibodies by an ELISA assay (see “Materials and Methods”). Virus shedding, specific IgG and IgA antibody, clinical score, and nasal secretion weight were determined, and the raw data for the wild-type virus and mutant tsl(B)-inoculated groups are given in Table 11. From such data the percentages of virus excretors, the percentage of responders, and the mean clinical score were calculated for the six groups. The data collected during this study are presented in summary form in Table 111.

Thirteen volunteers received saline. None showed any evidence of inapparent RS virus infection, and the clinical score of 0.9 provided a baseline for evaluation of the response of volunteers receiving virus-containing inocula. Twenty volunteers received wild-type virus, and 60% showed a significant immune response in terms of both specific IgG and IgA, or 90% in the case of either one or the other. Twenty percent shed virus. Forty-five percent of the group developed significant colds, and the mean clinical score for the group was 13.0. Despite the variability of response to infection of individuals (see Table II), the overall result is clear.

The groups that received mutants tsl9(A) or tsl9(B) showed much poorer responses. The immune responses (IgG and IgA) at 14% and 5 % , respectively, were far below the response in the group receiving the wild-type virus. The mean clinical scores and the percentages of colds were low, as were the virus excretion percentages. The virus recovered from mutant-inoculated volunteers retained the temperature sensitivity of the input virus. In each category the values for the tsl9(B) group were lower than those for the rsl9(A) group. These results are interpreted as the conse- quence of failure of the tsl9(A) and tsl9(B) mutants to replicate in the nasopharyngeal epithelium. The tsl9(B) mutant was the more deficient in this respect, as it might be expected since it was more temperature restricted.

The groups inoculated with the tsl(A) and tsl(B) mutants, on the other hand, showed good immune responses. The percentage immune response at 41 % for IgG and IgA and 68% overall in the tsl(B) group were the closest to those observed in the group inoculated with the wild-type virus. The mean clinical scores and the percentage of colds in the tsl(A) and tsl(B) groups were greatly reduced, with the mean clinical score of the tsl(B) group lower than that of the tsl(A) group. The frequency of virus recovery was not diminished in either group relative to the wild-type group. In all cases the virus recovered from volunteers inoculated with ts virus retained the temperature sensitivity of the input virus. These results suggest that the disease-

418 McKay et al.

TABLE 11. Wild-Type and Mutant tsl(B) Inoculated Groups: Clinical Scores, Nasal Secretion Weight, Virus Shedding, and Immune Response*

Nasal Specific IgG Specific IgA

Inoculurn Volunteer score weight OD ER OD ER Clinical secretion preserum preserum virus

isolation

rslB

WT 1 2 3 4 5 6 7 8 9

10 I 1 12 13 14 15 16 17 18 19 20

1 2 3 4 5 6 7 8 9

10 1 1 12 13 14 15 16 17 18 19 20 21 22

6" 0

35.5" 0.5 2

40a 37.5" 0.5

21a 12 7.5 1 0

25" 1 2" 3.5 7

43" 0 0 0.5 1.5 0.5 0 0 3.5 0 0 0 0 0 0 0 0

24" 1

10.5" 6" 0 0 0

4.46 5.89 3.31

85.74 0 0

75.53 98.93 0.84 8.05 2.03 0.76 0 0.58

22.28 5.39

20.35 3.96

110.21 0.46 0 5.14

22.37 52.52 0.04 2.69 9.43 0 5.97 1.84 0 0 0.54 2.73 0.16

39.35 0.11

14.40 1.78 0.66 0.83 0.73

1.05 0.90 1.36 1.05 1.10 0.67 0.93 1.14 0.98 0.56 0.44 0.47 0.66 0.65 0.70 0.82 0.93 0.52 0.58 ND 0.46 0.84 0.64 0.68 0.73 0.67 0.60 0.77 1.06 0.93 0.5 1 0.62 1.08 0.91 1.37 1.08 0.68 1.06 0.94 1.10 0.64 0.90

1.32 1.67 1.11

1.15 1.70 2.27 1.38 1.23 2.30 2.60 1.40 1 S O 1.42 1.76 1.42 1.61 1.72 1.90 NA 1.67 1.63 1.18 2.72 1.26 1.53 1.84 1.47 1.09 0.88 1.45 1.55 0.96 1.10 0.81 2.10 2.09 1.10 1.72 1.16 1.08 0.74

~

2.07

~

~

~

__ __ ~

__ ~

~

__ ~

__

__ __

~

__ __ __

~

~

__ ~

~

0.26 0.40 9.20 0.23 0.23 0.23 0.23 0.34 0.20 0.29 0.18 0.24 0.34 0.17 0.14 0.25 0.2 1 0.24 0.27 ND 0.18 0.16 0.18 0.14 0.13 0.15 0.15 0.15 0.18 0.20 0.28 0.24 0.16 0.39 0.14 0.12 0.12 0.22 0.20 0.14 0.49 0.30

1.70 1.73 1.92 1.61 1.40 1.91 2.45 1.48 1.65 1.67 1.88 1.25 1.88 1 .64 1.69 1.25 2.05 1.88 1.80 NA 1.50 1.40 1.22

1.77 2.33 1.80 1.53 1.10 1 S O 1.57 1 .oo 1.39 1 S O 1.43 2.91 1.82 1.40 3.60 2.70 1.20 1.40

~

__ ~

__

__ ~

~

~

~

~

~

__ __

~

~

~

~

2.78 ~

~

__ ~

~

~

~

~

__

__ ~

*The underlined values represent significant responses (see "Materials and Methods"). OD = optical density: ER = ELISA ratio; NA = not available; ND = not done. "Mild to moderate cold.


TABLE 111. The Summarised Outcome of Assessment in Adult Volunteers of the Irnmunogenicity and Pathogenic Potential of the RSS-2 Strain of RS Virus and Four ts Mutant Derivatives

No. of volunteers

13 20 20 22 22 22

Inoculum ( I .OOo Pfu)

(Saline) W ild-type rs 1 (A) rsl(B) fsl9(A) fs I91B)

Percent virus

excretion

Nil" 20 25 32 9 5

Percent immune response IgA or IgA and

IgG IgA IgG IgG

Nil Nil Nil Nil 90 80 70 60 55 4s 35 25 68 59 50 41 18 18 18 14 I4 9 9 5

Percent coids

Nil 45 I0 14 9 5

Mean clinical score

0.9 13.0 5.3 2.2 6.6 1.1

"Nil < 7%.

producing potential of the tsl(A) and tsl(B) mutants has been reduced without a diminution in replicative ability. The more modified tsl(B) has proceded further along this path than tsI(A), yet it still retains an immunising capacity not much inferior to that of the wild-type virus. This mutant shows attributes appropriate to a vaccine: i.e. immunogenicity, reduced pathogenicity, retention of replicative ability in the nasopharynx, and genetic stability.

DISCUSSION

Single ts mutants whose multiplication is restricted in diploid human foetal lung cells at 39°C and double ts mutants whose multiplication is restricted in the same cells at 38°C have been obtained following chemical mutagenesis. Mutants were isolated sequentially using mutagens with different chemical specificities to increase the probability of inducing mutations at independent sites in the RS virus genome, thereby reducing the possibility of loss of temperature sensitivity by direct reversion. The results of the trials in human volunteers described above suggest that this aim was achieved since the virus recovered from volunteers after multiplication in the nasopharynx for 7 days retained its temperature-sensitive phenotype.

The assessment of these mutants was carried out in adult volunteers with varying levels of pre-existing secretory, humoral, and cell-mediated immunity. Nevertheless clinical signs and symptoms were induced in nearly two-thirds of the group receiving the witd-type virus. Among other things this showed that the limited number of passages of the RSS-2 strain in MRC-5 cells required for the production and propagation of the mutants had not by itself diminished the pathogenic potential of the virus. The results of the trials show that administration of a temperature-restricted live RS virus can produce a secondary immune response almost equivalent to that produced by wild-type virus but with markedly reduced clinical signs and symptoms. It is also dear from the results of the volunteer triais that the ts mutants with the RNA-positive phenotypes tsl(A) and tsl(B) were more effective as a live vaccine than the more completely temperature-restricted tsl9(A) or tsl9(B).

Now that it has been shown that the wild-type RSS-2 strain produces moderate clinical disease in adults it is feasible to proceed to virus challenge experiments to determine whether the residual immunity in adults can be boosted to a protective level by administration of the tsl(B) mutant.

It is unlikely that the performance of these variants would be comparable in non-immune infants, the target population for protective immunisation, particularly

420 McKay et al.

with regard to genetic stability in view of earlier experience with the A2 strain mutants [Kim et al., 1973; Hodes et al., 19741. Also the residual pathogenicity of even the most modified mutant, tsl(B), would not be acceptable. Nevertheless the results described in this paper are encouraging, in that the properties of the virus have been modified in the right direction, and further modification of the tsl(B) mutant will be attempted. For example, a further reduction in the temperature threshold of mutant tsl(B) might be sufficient to eliminate residual pathogenicity. Although the production of a fusion-protein-based RS virus vaccine by genetic engineering tech- niques is an attractive prospect [Pringle, 19871, not enough is known yet about the precise nature of protective immunity to RS virus infection in infancy to be certain that this approach will succeed. From animal experiments it appears that a balanced immune response is a prerequisite for induction of protective immunity [Murphy et al., 19861. This may be easier to achieve by means of a live virus vaccine.

Irrespective of the future development of the tsl(B) mutant, these trials have served to provide a database that may be useful for comparative assessment of the performance of any candidate RS virus vaccine that may be produced by other means in the future. A detailed analysis of the immune response of these volunteers to individual RS virus proteins will be published separately (Watt et al., unpublished data). Analysis of the properties of the tsl(A) and tsl(B) variants may identify the determinants of virulence.

ACKNOWLEDGMENTS

This work was supported by grants from the Medical Research Council and the Vaccine Development Programme of the World Health Organization. We would like to thank Dr. I. Barrow, Mrs. A. Dalton, and Miss J. Dunning for the clinical observations and the volunteers for their willing cooperation.

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Immunogenicity and pathogenicity of temperature-sensitive modified respiratory syncytial virus in...

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