901 to the liposome preparation to give a final of 200 l1-g/ml viral subunit and 0.5 g/dl lipid, composition and this mixture was sonicated for a further 15 minutes.
subunits were added
RESULTS
Examination of the original liposome preparation in the electron microscope by the negative-staining technique showed that the vast majority of the lipid bodies were small unilamellar structures with a few larger multilamellar liposomes (fig. 1 a). Most of the liposomes were in the size range 20-100 nm. Examination of the influenza-virus-subunit preparation revealed only the typical star and cartwheel forms associated with haemagglutinin and neuraminidase subunits (fig. lb). No trace of viral membrane could be found in the subunit specimens. When the sonicate of viral subunits and liposomes was examined, only a few viral subunits remained in the background. Most of the subunits were now arrayed on the liposomes (fig. 2a) to give an appearance not unlike that of whole influenza virus (fig. 2b). Areas were recognised where neuraminidase rather than haemagglutinin subunits were attached, although these were in the minority (fig. 2c). These viral liposomes were stored for several months at 4°C and electron microscopy showed then to be stable structures. The viral liposomes were examined by immune electron microscopy, and it was found that they were aggregated into large complexes by the addition of hyperimmune influenza-A antiserum.
DISCUSSION
Previous studies of the use of liposomes have emphasised the entrapment of drugs, enzymes, and antigens within the internal aqueous compartment of the multilamellar lipid body.1-3 We have reversed this approach by attaching the subunits to the surface of a unilamellar liposome and have avoided entrapment by the use of pre-formed liposomes. The orientation of the subunits on the liposomes, and in particular the orientation of neuraminidase which is an asymmetric structure, leads us to believe that the subunits are in fact arrayed on the liposome in the same manner as they are on the virus particle. Further, the degree to which the viral liposomes can be aggregated by the use of anti-influenza serum indicates that most of the original binding sites are still available for antibody attachment. The hydrophobic bonding which retains the haemagglutinin and neuraminidase in the influenza virus envelope probably produces the same orientation of the subunits on the liposome surface as on the original virus particle. In view of the structural similarities between the viral liposomes and the original virus particles, we have called the new structures virosomes. The artificial virus particles, virosomes, described here may help to solve some of the problems of influenza vaccination. Although whole inactivated influenza virus is
good immunogen, it is also pyrogenic. However highly purified influenza virus subunits, while not pyrogenic,9 are not very satisfactory immunogens.10 Preliminary work suggests that virosomes are both immunogenic and non-pyrogenic, and manipulation of the base on which the subunits are presented could well be a method of ima
proving adjuvanticity genicity.
in the absence of increased pyro-
We thank Mr J. Rowe and Miss J. Cole for their technical assistT.D.H. is a recipient of a CASE award from the Science Research Council. Requests for reprints should be addressed to J.D.A.
ance.
REFERENCES
1. Gregoriadis, G. FEBS Letts, 1973, 56, 292. 2. Gregoriadis, G., Ryman, B. E. Biochem. J.1972, 129, 123. 3. Allison, A. C., Gregoriadis, G. Nature, 1974, 252, 252. 4. Heath, T. D., Edwards, D. C., Ryman, B. E. Unpublished. 5. Bangham, A. D., Hill, M. W., Miller, N. G. A. in Methods in Membrane Biology (edited by E. D. Korn); vol. 1, p. 1. London, 1974. 6. Lauer, W. G., Valentine, R. C. Virology, 1969, 38, 105. 7. Skehel, J. J., Schild, G. C. ibid. 1971, 44, 396. 8. Almeida, J. D., Hoyle, L. Micron, 1972, 3, 306. 9. Webster, R. G., Lauer, W. G. J. Immun. 1966, 96, 596. 10. Jennings, R. Brand, C. M., McLaren, C., Shepherd, L., Potter, C. W. Med. microbiol. Immun. 1974, 160, 295.
TRANSFER FACTOR IN HODGKIN’S DISEASE C. G. ALEXOPOULOS A. J. BELLINGHAM*
R. P. NG C. J. MORAN
Department of Clinical Hœmatology, University College Hospital Medical School, London WC1E 6HX controlled study, six patients with stage-iv Hodgkin’s disease were given transfer factor (T.F.) prepared from patients with Hodgkin’s disease in long remission. There was an apparent increase in cell-mediated immune responses as evidenced by a significant increase in the recipients’ lymphocyte responses to phytohæmagglutinin stimulation. Three out of six patients converted to positive delayed-hypersensitivity tests. These three all had the nodular sclerosing In
Summary
a
type of Hodgkin’s disease. These results warrant the further investigation of the use of Hodgkin’s disease-specific T.F. as a therapeutic agent in this condition. INTRODUCTION
known as transfer factor (T.F.) can transfer specific cell-mediated immunity between normal individuals.’ Any therapeutic value will lie in its ability to transfer or boost cellular immunity in patients, since immune depression or tolerance may play a significant part in the pathological process. Immune depression in Hodgkin’s disease is well estabwith depressed delayed-type hypersensitivity and decreased lymphocyte response to phytohaemagglutimn (P.H.A.).2 Of thirty-five recorded attempts at transfer in Hodgkin’s disease by three groups of workers using normal donors, there was only one successful transfer of tuberculin sensitivity.3-5 Because of this apparent resistance in Hodgkin’s disease to transfer from normal donors, it seemed more logical to use disease-specific T.F. from patients in longterm remission, and to see if this was more effective as a potential therapeutic agent. THE
lymphocyte
extract
lished,
PATIENTS AND METHODS
Twelve
patients
"Present address:
with stage-iv
Hodgkin’s
disease from the
Department of Hæmatology, University of Liverpool.
902
University College Hospital lymphoma clinic were divided into pairs. Studies were performed either before treatment started, or at least 4 weeks after chemotherapy finished.
six matched
TABLE
H)—RESPONSE
RECEIVING
T.F.,
TO
P.H.A. IN RECIPIENTS BEFORF AND AFTIR
AND IN CONTROI. PATIENTS WHFRF THF STI’DIFS vFR1
REPEATED ONE MONTH APART WITHOUT THF
SUBJFCTS
BEING GI1FN A",
T.F.
Preparation of T.F. Donors were patients with Hodgkin’s disease in remission for more than 5 years. They all gave positive skin tests to candida and tuberculin. Leucocytes were harvested by leucopheresis, each donor giving 5 x 109 lymphocytes (IT.F. unit is equivalent to 109 lymphocytes). An average of 3 units of T.F. were given to each of the six recipients. The number of lymphocytes removed from each donor constituted less than 0-5% of the total lymphocyte population and no ill-effects have been observed over one year’s observation. Skin Tests Skin testing was carried out before each injection of T.F. and repeated 3-7 days after. Induration of more than 5 mm diameter at 48 h was read as positive, 2-4 mm as equivocal,
subunits were added
RESULTS
Examination of the original liposome preparation in the electron microscope by the negative-staining technique showed that the vast majority of the lipid bodies were small unilamellar structures with a few larger multilamellar liposomes (fig. 1 a). Most of the liposomes were in the size range 20-100 nm. Examination of the influenza-virus-subunit preparation revealed only the typical star and cartwheel forms associated with haemagglutinin and neuraminidase subunits (fig. lb). No trace of viral membrane could be found in the subunit specimens. When the sonicate of viral subunits and liposomes was examined, only a few viral subunits remained in the background. Most of the subunits were now arrayed on the liposomes (fig. 2a) to give an appearance not unlike that of whole influenza virus (fig. 2b). Areas were recognised where neuraminidase rather than haemagglutinin subunits were attached, although these were in the minority (fig. 2c). These viral liposomes were stored for several months at 4°C and electron microscopy showed then to be stable structures. The viral liposomes were examined by immune electron microscopy, and it was found that they were aggregated into large complexes by the addition of hyperimmune influenza-A antiserum.
DISCUSSION
Previous studies of the use of liposomes have emphasised the entrapment of drugs, enzymes, and antigens within the internal aqueous compartment of the multilamellar lipid body.1-3 We have reversed this approach by attaching the subunits to the surface of a unilamellar liposome and have avoided entrapment by the use of pre-formed liposomes. The orientation of the subunits on the liposomes, and in particular the orientation of neuraminidase which is an asymmetric structure, leads us to believe that the subunits are in fact arrayed on the liposome in the same manner as they are on the virus particle. Further, the degree to which the viral liposomes can be aggregated by the use of anti-influenza serum indicates that most of the original binding sites are still available for antibody attachment. The hydrophobic bonding which retains the haemagglutinin and neuraminidase in the influenza virus envelope probably produces the same orientation of the subunits on the liposome surface as on the original virus particle. In view of the structural similarities between the viral liposomes and the original virus particles, we have called the new structures virosomes. The artificial virus particles, virosomes, described here may help to solve some of the problems of influenza vaccination. Although whole inactivated influenza virus is
good immunogen, it is also pyrogenic. However highly purified influenza virus subunits, while not pyrogenic,9 are not very satisfactory immunogens.10 Preliminary work suggests that virosomes are both immunogenic and non-pyrogenic, and manipulation of the base on which the subunits are presented could well be a method of ima
proving adjuvanticity genicity.
in the absence of increased pyro-
We thank Mr J. Rowe and Miss J. Cole for their technical assistT.D.H. is a recipient of a CASE award from the Science Research Council. Requests for reprints should be addressed to J.D.A.
ance.
REFERENCES
1. Gregoriadis, G. FEBS Letts, 1973, 56, 292. 2. Gregoriadis, G., Ryman, B. E. Biochem. J.1972, 129, 123. 3. Allison, A. C., Gregoriadis, G. Nature, 1974, 252, 252. 4. Heath, T. D., Edwards, D. C., Ryman, B. E. Unpublished. 5. Bangham, A. D., Hill, M. W., Miller, N. G. A. in Methods in Membrane Biology (edited by E. D. Korn); vol. 1, p. 1. London, 1974. 6. Lauer, W. G., Valentine, R. C. Virology, 1969, 38, 105. 7. Skehel, J. J., Schild, G. C. ibid. 1971, 44, 396. 8. Almeida, J. D., Hoyle, L. Micron, 1972, 3, 306. 9. Webster, R. G., Lauer, W. G. J. Immun. 1966, 96, 596. 10. Jennings, R. Brand, C. M., McLaren, C., Shepherd, L., Potter, C. W. Med. microbiol. Immun. 1974, 160, 295.
TRANSFER FACTOR IN HODGKIN’S DISEASE C. G. ALEXOPOULOS A. J. BELLINGHAM*
R. P. NG C. J. MORAN
Department of Clinical Hœmatology, University College Hospital Medical School, London WC1E 6HX controlled study, six patients with stage-iv Hodgkin’s disease were given transfer factor (T.F.) prepared from patients with Hodgkin’s disease in long remission. There was an apparent increase in cell-mediated immune responses as evidenced by a significant increase in the recipients’ lymphocyte responses to phytohæmagglutinin stimulation. Three out of six patients converted to positive delayed-hypersensitivity tests. These three all had the nodular sclerosing In
Summary
a
type of Hodgkin’s disease. These results warrant the further investigation of the use of Hodgkin’s disease-specific T.F. as a therapeutic agent in this condition. INTRODUCTION
known as transfer factor (T.F.) can transfer specific cell-mediated immunity between normal individuals.’ Any therapeutic value will lie in its ability to transfer or boost cellular immunity in patients, since immune depression or tolerance may play a significant part in the pathological process. Immune depression in Hodgkin’s disease is well estabwith depressed delayed-type hypersensitivity and decreased lymphocyte response to phytohaemagglutimn (P.H.A.).2 Of thirty-five recorded attempts at transfer in Hodgkin’s disease by three groups of workers using normal donors, there was only one successful transfer of tuberculin sensitivity.3-5 Because of this apparent resistance in Hodgkin’s disease to transfer from normal donors, it seemed more logical to use disease-specific T.F. from patients in longterm remission, and to see if this was more effective as a potential therapeutic agent. THE
lymphocyte
extract
lished,
PATIENTS AND METHODS
Twelve
patients
"Present address:
with stage-iv
Hodgkin’s
disease from the
Department of Hæmatology, University of Liverpool.
902
University College Hospital lymphoma clinic were divided into pairs. Studies were performed either before treatment started, or at least 4 weeks after chemotherapy finished.
six matched
TABLE
H)—RESPONSE
RECEIVING
T.F.,
TO
P.H.A. IN RECIPIENTS BEFORF AND AFTIR
AND IN CONTROI. PATIENTS WHFRF THF STI’DIFS vFR1
REPEATED ONE MONTH APART WITHOUT THF
SUBJFCTS
BEING GI1FN A",
T.F.
Preparation of T.F. Donors were patients with Hodgkin’s disease in remission for more than 5 years. They all gave positive skin tests to candida and tuberculin. Leucocytes were harvested by leucopheresis, each donor giving 5 x 109 lymphocytes (IT.F. unit is equivalent to 109 lymphocytes). An average of 3 units of T.F. were given to each of the six recipients. The number of lymphocytes removed from each donor constituted less than 0-5% of the total lymphocyte population and no ill-effects have been observed over one year’s observation. Skin Tests Skin testing was carried out before each injection of T.F. and repeated 3-7 days after. Induration of more than 5 mm diameter at 48 h was read as positive, 2-4 mm as equivocal,
